Compounds for preparing immunological adjuvant

ABSTRACT

The present invention provides methods for preparing TLR-4 receptor agonist E6020: 
     
       
         
         
             
             
         
       
     
     and stereoisomers thereof, which compounds are useful as an immunological adjuvants when co-administered with antigens such as vaccines for bacterial and viral diseases. Also provided are synthetic intermediates.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to U.S.Provisional Application Ser. No. 60/695,324, filed Jun. 30, 2005, whichis incorporated herein by reference.

BACKGROUND OF THE INVENTION

Generally, vaccines have proven to be successful methods for theprevention of infectious diseases. Generally, they are cost effective,and do not induce antibiotic resistance to the target pathogen or affectnormal flora present in the host. In many cases, such as when inducinganti-viral immunity, vaccines can prevent a disease for which there areno viable curative or ameliorative treatments available.

Vaccines function by triggering the immune system to mount a response toan agent, or antigen, typically an infectious organism or a portionthereof that is introduced into the body in a non-infectious ornon-pathogenic form. Once the immune system has been “primed” orsensitized to the organism, later exposure of the immune system to thisorganism as an infectious pathogen results in a rapid and robust immuneresponse that destroys the pathogen before it can multiply and infectenough cells in the host organism to cause disease symptoms.

The agent, or antigen, used to prime the immune system can be the entireorganism in a less infectious state, known as an attenuated organism, orin some cases, components of the organism such as carbohydrates,proteins or peptides representing various structural components of theorganism.

In many cases, it is necessary to enhance the immune response to theantigens present in a vaccine in order to stimulate the immune system toa sufficient extent to make a vaccine effective, i.e., to conferimmunity. Many protein and most peptide and carbohydrate antigens,administered alone, do not elicit a sufficient antibody response toconfer immunity. Such antigens need to be presented to the immune systemin such a way that they will be recognized as foreign and will elicit animmune response. To this end, additives (adjuvants) have been devisedwhich immobilize antigens and stimulate the immune response.

The best known adjuvant, Freund's complete adjuvant, consists of amixture of mycobacteria in an oil/water emulsion. Freund's adjuvantworks in two ways: first, by enhancing cell and humoral-mediatedimmunity, and second, by blocking rapid dispersal of the antigenchallenge (the “depot effect”). However, due to frequent toxicphysiological and immunological reactions to this material, Freund'sadjuvant cannot be used in humans.

Another molecule that has been shown to have immunostimulatory oradjuvant activity is endotoxin, also known as lipopolysaccharide (LPS).LPS stimulates the immune system by triggering an “innate” immuneresponse—a response that has evolved to enable an organism to recognizeendotoxin (and the invading bacteria of which it is a component) withoutthe need for the organism to have been previously exposed. While LPS istoo toxic to be a viable adjuvant, molecules that are structurallyrelated to endotoxin, such as monophosphoryl lipid A (“MPL”) are beingtested as adjuvants in clinical trials. Both LPS and MPL have beendemonstrated to be agonists to the human toll-like recEptor-4 (TLR-4).Currently, however, the only FDA-approved adjuvant for use in humans isaluminum salts (Alum) which are used to “depot” antigens byprecipitation of the antigens. Alum also stimulates the immune responseto antigens.

Accordingly, there is a need to develop synthetic methods for preparingcompounds which can be co-administered with antigens in order tostimulate the immune system to generate a more robust antibody responseto the antigen than would be seen if the antigen were injected alone orwith Alum.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for synthesizingthe TLR-4 receptor agonist E6020 having the structure:

In another aspect, the invention encompasses methods for synthesizingany stereoisomer of E6020. Thus there is provided herein a syntheticintermediates for preparing a compound having the structure:

These compounds are useful as immunological adjuvants whenco-administered with antigens such as vaccines for bacterial and viraldiseases. The present invention also provides synthetic intermediatesuseful for preparing E6020 and stereoisomers thereof.

BRIEF DESCRIPTIONS OF THE FIGURES

FIG. 1 depicts the structure of crystalline ER-8016158.

FIG. 2 is the packing diagram along the a-axis which shows the bestdiagram of the hydrogen bonding within the ER-806158 crystal, dottedlines.

FIG. 3 depicts the Powder X-ray Diffraction (PXRD) pattern ofcrystalline ER-806158.

FIG. 4 shows the DSC thermograms of crystalline ER-806158.

FIG. 5 shows the infrared spectrum of crystalline ER-806158.

DEFINITIONS

In accordance with the present invention and as used herein, thefollowing terms are defined with the following meanings, unlessexplicitly stated otherwise.

Certain compounds disclosed in the present invention, and definitions ofspecific functional groups are also described in more detail below. Forpurposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in “OrganicChemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999,the entire contents of which are incorporated herein by reference.Furthermore, it will be appreciated by one of ordinary skill in the artthat the synthetic methods, as described herein, utilize a variety ofprotecting groups. By the term “protecting group”, has used herein, itis meant that a particular functional moiety, e.g., O, S, P, or N, istemporarily blocked so that a reaction can be carried out selectively atanother reactive site in a multifunctional compound. In preferredembodiments, a protecting group reacts selectively in good yield to givea protected substrate that is stable to the projected reactions; theprotecting group must be selectively removed in good yield by readilyavailable, preferably nontoxic reagents that do not attack the otherfunctional groups; the protecting group forms an easily separablederivative (more preferably without the generation of new stereogeniccenters); and the protecting group has a minimum of additionalfunctionality to avoid further sites of reaction. As detailed herein,oxygen, sulfur, nitrogen, phosphorous, and carbon protecting groups maybe utilized.

For example, in certain embodiments, as detailed herein, certainexemplary oxygen protecting groups are utilized. These oxygen protectinggroups include, but are not limited to methyl ethers, substituted methylethers (e.g., MOM (methoxymethyl ether), MTM (methylthiomethyl ether),BOM (benzyloxymethyl ether), PMBM (p-methoxybenzyloxymethyl ether), toname a few), substituted ethyl ethers, substituted benzyl ethers, silylethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilylether), TIPS(triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzylsilyl ether, TBDPS (t-butyldiphenyl silyl ether)), esters (e.g.,formate, acetate, benzoate (Bz), trifluoroacetate, dichloroacetate, toname a few), carbonates, cyclic acetals and ketals. Protecting groupsfor phosphite oxygens and phosphate oxgens include, for example, alkylphosphates/phosphites such as: methyl, ethyl; isopropyl; t-butyl;cyclohexyl; 1-adamantyl; and 2-trimethylsilylprop-2-enyl; alkenylphosphates/phospites such as ethenyl and allyl; 2-substituted ethylphosphates/phosphites such as: 2-cyanoethyl, 2-cyano-1,1-dimethylethyl,2-(trimethylsilyl)ethyl, 2-(4-nitrophenyl)ethyl,2-(phenylsulfonyl)ethyl, and 2-(benzylsulfonyl)ethyl; haloethylphosphates/phosphites such as: 2,2,2-trichloroethyl,2,2,2-trichloro-1,1-dimethylethyl, 2,2,2-tribromoethyl,2,3-dibromopropyl, benzyl phosphates/phosphates such as: benzyl;4-nitrobenzyl, 4-chlorobenzyl; 1-oxido-4-methoxy-2-picolyl,fluorenyl-9-methyl, 5-benzisoxazolylmethylene, (C₆H₅)₂C═; and phenylphosphates/phosphites such as: phenyl; 4-nitrophenyl, and4-chlorophenyl; and silyl phosphates/phosphites such as: trimethylsilyl.

In certain other exemplary embodiments, nitrogen protecting groups areutilized. These nitrogen protecting groups may be monovalent or divalentprotecting groups such as, but are not limited to, carbamates (includingmethyl, ethyl and substituted ethyl carbamates (e.g., Troc), to name afew) amides, cyclic imide derivatives, N-Alkyl and N-Aryl amines, iminederivatives, and enamine derivatives, to name a few. Amine protectinggroups such as Cbz, Boc, Fmoc, TROC, TMS-ethylcarbonyl,cyanoethylcarbonyl, allyloxycarbonyl or (C₆H₅)₂C═ (diphenylmethylene)may also be mentioned. Certain other exemplary protecting groups aredetailed herein, however, it will be appreciated that the presentinvention is not intended to be limited to these protecting groups;rather, a variety of additional equivalent protecting groups can bereadily identified using the above criteria and utilized in the presentinvention. Additionally, a variety of protecting groups are described in“Protective Groups in Organic Synthesis” Third Ed. Greene, T. W. andWuts, P. G., Eds., John Wiley & Sons, New York: 1999, the entirecontents of which are hereby incorporated by reference.

It is understood that the compounds, as described herein, may besubstituted with any number of substituents or functional moieties. Ingeneral, the term “substituted” whether preceded by the term“optionally” or not, and substituents contained in formulas of thisinvention, refer to the replacement of hydrogen radicals in a givenstructure with the radical of a specified substituent. When more thanone position in any given structure may be substituted with more thanone substituent selected from a specified group, the substituent may beeither the same or different at every position. As used herein, the term“substituted” is contemplated to include all permissible substituents oforganic compounds. In a broad aspect, the permissible substituentsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and non-aromatic, carbon and heteroatomsubstituents of organic compounds. For purposes of this invention,heteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valencies of the heteroatoms. Furthermore, this invention isnot intended to be limited in any manner by the permissible substituentsof organic compounds. Combinations of substituents and variablesenvisioned by this invention are preferably those that result in theformation of stable compounds useful in the treatment and prevention,for example of disorders, as described generally above. Examples ofsubstituents include, but are not limited to, halo substituents, e.g. F;Cl; Br; or I; a hydroxyl group; a C₁-C₆ alkoxy group, e.g, —OCH₃,—OCH₂CH₃, or —OCH(CH₃)₂; a C₁-C₆ haloalkyl group, e.g., —CF₃; —CH₂CF₃;or —CHCl₂; C₁-C₆ alkylthio; amino; mono and dialkyl amino groups; —NO₂;—CN; a sulfate group, and the like. Additional examples of generallyapplicable substituents are illustrated by the specific embodimentsshown in the Examples that are described herein.

The term “stable”, as used herein, preferably refers to compounds whichpossess stability sufficient to allow manufacture and which maintain theintegrity of the compound for a sufficient period of time to be detectedand preferably for a sufficient period of time to be useful for thepurposes detailed herein.

As used herein, the term “alkyl” includes straight and branched alkylgroups. An analogous convention applies to other generic terms such as“alkenyl”, “alkynyl” and the like. Furthermore, as used herein, theterms “alkyl”, “alkenyl”, “alkynyl” and the like encompass bothsubstituted and unsubstituted groups. In certain embodiments, as usedherein, “lower alkyl” is used to indicate those alkyl groups (cyclic,acyclic, substituted, unsubstituted, branched or unbranched) having 1-6carbon atoms. In other embodiments, C₁₋₄, C₂₋₄, C₁₋₃ or C₃₋₆ alkyl oralkenyl are preferred.

In certain embodiments, the alkyl, alkenyl and alkynyl groups employedin the invention contain 1-20 aliphatic carbon atoms for alkyl groupsand 2-20 carbon atoms for alkenyl and alkynyl groups. In certain otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain 1-15 aliphatic carbon atoms. In yet other embodiments,the alkyl, alkenyl, and alkynyl groups employed in the invention contain1-8 aliphatic carbon atoms. In still other embodiments, the alkyl,alkenyl, and alkynyl groups employed in the invention contain 1-6aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups employed in the invention contain 1-4 carbon atoms.Illustrative aliphatic groups thus include, but are not limited to, forexample, methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, sec-butyl,isobutyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl,n-hexyl, sec-hexyl, moieties and the like, which again, may bear one ormore substituents. Alkenyl groups include, but are not limited to, forexample, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and thelike. Representative alkynyl groups include, but are not limited to,ethynyl, 2-propynyl (propargyl), 1-propynyl and the like.

The term “alicyclic”, as used herein, refers to compounds which combinethe properties of aliphatic and cyclic compounds and include but are notlimited to cyclic, or polycyclic aliphatic hydrocarbons and bridgedcycloalkyl compounds, which are optionally substituted with one or morefunctional groups. As will be appreciated by one of ordinary skill inthe art, “alicyclic” is intended herein to include, but is not limitedto, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties, which areoptionally substituted with one or more functional groups. Illustrativealicyclic groups thus include, but are not limited to, for example,cyclopropyl, —CH₂-cyclopropyl, cyclobutyl, —CH₂-cyclobutyl, cyclopentyl,—CH₂-cyclopentyl-n, cyclohexyl, —CH₂-cyclohexyl, cyclohexenylethyl,cyclohexanylethyl, norborbyl moieties and the like, which again, maybear one or more substituents.

The term “alkoxy” (or “alkyloxy”), or “thioalkyl” as used herein refersto an alkyl or cycloalkyl group, as previously defined, attached to theparent molecular moiety through an oxygen atom or through a sulfur atom.In certain embodiments, the alkyl or cycloalkyl group contains 1-20aliphatic or alicyclic carbon atoms. In certain other embodiments, thealkyl or cycloalkyl group contains 1-10 aliphatic or alicyclic carbonatoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groupsemployed in the invention contain 1-8 aliphatic or alicyclic carbonatoms. In still other embodiments, the alkyl group contains 1-6aliphatic or alicyclic carbon atoms. In yet other embodiments, the alkylgroup contains 1-4 aliphatic or alicyclic carbon atoms. Examples ofalkoxy, include but are not limited to, methoxy, ethoxy, propoxy,isopropoxy, n-butoxy, tert-butoxy, neopentoxy and n-hexoxy. Examples ofthioalkyl include, but are not limited to, methylthio, ethylthio,propylthio, isopropylthio, n-butylthio, and the like.

The term “alkylamino” refers to a group having the structure —NHR′wherein R′ is alkyl or cycloalkyl, as defined herein. The term“dialkylamino” refers to a group having the structure —N(R′)₂, whereineach occurrence of R′ is independently alkyl or cycloalkyl, as definedherein. The term “aminoalkyl” refers to a group having the structureNH₂R′—, wherein R′ is alkyl or cycloalkyl, as defined herein. In certainembodiments, the alkyl group contains 1-20 aliphatic or alicyclic carbonatoms. In certain other embodiments, the alkyl or cycloalkyl groupcontains 1-10 aliphatic or alicyclic carbon atoms. In yet otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain 1-8 aliphatic or alicyclic carbon atoms. In stillother embodiments, the alkyl or cycloalkyl group contains 1-6 aliphaticor alicyclic carbon atoms. In yet other embodiments, the alkyl orcycloalkyl group contains 1-4 aliphatic or alicyclic carbon atoms.Examples of alkylamino include, but are not limited to, methylamino,ethylamino, iso-propylamino and the like.

In general, the terms “aryl” and “heteroaryl”, as used herein, refer tostable mono- or polycyclic, heterocyclic, polycyclic, andpolyheterocyclic unsaturated moieties having preferably 3-14 carbonatoms, each of which may be substituted or unsubstituted. It will alsobe appreciated that aryl and heteroaryl moieties, as defined herein maybe attached via an alkyl or heteroalkyl moiety and thus also include-(alkyl)aryl, -(heteroalkyl)aryl, -(heteroalkyl)aryl, and-(heteroalkyl)heteroaryl moieties. Thus, as used herein, the phrases“aryl or heteroaryl” and “aryl, heteroaryl, -(alkyl)aryl,-(heteroalkyl)aryl, -(heteroalkyl)aryl, and (heteroalkyl)heteroaryl” areinterchangeable. Substituents include, but are not limited to, any ofthe previously mentioned substitutents, i.e., the substituents recitedfor aliphatic moieties, or for other moieties as disclosed herein,resulting in the formation of a stable compound. In certain embodimentsof the present invention, “aryl” refers to a mono- or bicycliccarbocyclic ring system having one or two aromatic rings including, butnot limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyland the like. In certain embodiments of the present invention, the term“heteroaryl”, as used herein, refers to a cyclic aromatic radical havingfrom five to ten ring atoms of which one ring atom is selected from S, Oand N; zero, one or two ring atoms are additional heteroatomsindependently selected from S, O and N; and the remaining ring atoms arecarbon, the radical being joined to the rest of the molecule via any ofthe ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl,pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl,thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, and the like.

It will be appreciated that aryl and heteroaryl groups (includingbicyclic aryl groups) can be unsubstituted or substituted, whereinsubstitution includes replacement of one or more of the hydrogen atomsthereon independently with any one or more of the substituents generallydescribed above. Additional examples of generally applicablesubstituents are illustrated by the specific embodiments shown in theExamples that are described herein.

The term “cycloalkyl”, as used herein, refers specifically to groupshaving three to seven, preferably three to ten carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the caseof other alicyclic, heteroalicyclic or heterocyclic moieties, mayoptionally be substituted with one or more of the substituents generallydescribed above. An analogous convention applies to other generic termssuch as “cycloalkenyl”, “cycloalkynyl” and the like. Additionally, itwill be appreciated that any of the alicyclic or heteroalicyclicmoieties described above and herein may comprise an aryl or heteroarylmoiety fused thereto. Additional examples of generally applicablesubstituents are illustrated by the specific embodiments shown in theExamples that are described herein.

The term “heteroaliphatic”, as used herein, refers to aliphatic moietiesin which one or more carbon atoms in the main chain have beensubstituted with a heteroatom. Thus, a heteroaliphatic group refers toan aliphatic chain which contains one or more oxygen, sulfur, nitrogen,phosphorus or silicon atoms, e.g., in place of carbon atoms.Heteroaliphatic moieties may be branched or linear unbranched. Incertain embodiments, heteroaliphatic moieties are substituted byindependent replacement of one or more of the hydrogen atoms thereonwith one or more of the substituents generally described above.Additional examples of generally applicable substituents are illustratedby the specific embodiments shown in the Examples that are describedherein.

The term “heteroalicyclic”, as used herein, refers to compounds whichcombine the properties of heteroaliphatic and cyclic compounds andinclude but are not limited to saturated and unsaturated mono- orpolycyclic heterocycles such as morpholino, pyrrolidinyl, furanyl,thiofuranyl, pyrrolyl etc., which are optionally substituted with one ormore functional groups, as defined herein.

Additionally, it will be appreciated that any of the alicyclic orheteroalicyclic moieties described above and herein may comprise an arylor heteroaryl moiety fused thereto. Additional examples of generallyapplicable substituents are illustrated by the specific embodimentsshown in the Examples that are described herein.

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine, chlorine, bromine and iodine.

The term “haloalkyl” denotes an alkyl group, as defined above, havingone, two, or three halogen atoms attached thereto and is exemplified bysuch groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term “heterocycloalkyl” or “heterocycle”, as used herein, refers toa non-aromatic 5-, 6- or 7-membered ring or a polycyclic group,including, but not limited to a bi- or tri-cyclic group comprising fusedsix-membered rings having between one and three heteroatomsindependently selected from oxygen, sulfur and nitrogen, wherein (i)each 5-membered ring has 0 to 1 double bonds and each 6-membered ringhas 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may beoptionally be oxidized, (iii) the nitrogen heteroatom may optionally bequaternized, and (iv) any of the above heterocyclic rings may be fusedto a substituted or unsubstituted aryl or heteroaryl ring.Representative heterocycles include, but are not limited to,pyrrolidinyl, pyrazolinyl, pyrazolidiny1, imidazolinyl, imidazolidinyl,piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. In certainembodiments, a “substituted heterocycloalkyl or heterocycle” group isutilized and as used herein, refers to a heterocycloalkyl or heterocyclegroup, as defined above, substituted by the independent replacement ofone or more of the hydrogen atoms thereon with one or more of thesubstituents generally described above. Additional examples or generallyapplicable substituents are illustrated by the specific embodimentsshown in the Examples that are described herein.

As used herein, the terms “aliphatic”, “heteroaliphatic”, “alkyl”,“alkenyl”, “alkynyl”, “heteroalkyl”, “heteroalkenyl”, “heteroalkynyl”,and the like encompass substituted and unsubstituted, saturated andunsaturated, and linear and branched groups. Similarly, the terms“alicyclic”, “heteroalicyclic”, “heterocycloalkyl”, “heterocycle” andthe like encompass substituted and unsubstituted, and saturated andunsaturated groups. Additionally, the terms “cycloalkyl”,“cycloalkenyl”, “cycloalkynyl”, “heterocycloalkyl”,“heterocycloalkenyl”, “heterocycloalkynyl”, “aryl”, “heteroaryl” and thelike encompass both substituted and unsubstituted groups.

Further, E6020 contains asymmetric carbon atoms and hence can exist asstereoisomers, both enantiomers and diastereomers. One of ordinary skillin the art will recognize that the inventive method may be adapted tothe preparation of any of all possible stereoisomers of E6020. While theexamples provided herein disclose the preparation of a particularisomer, methods for preparing other stereoisomers of E6020 areconsidered to fall within the scope of the present invention.

DETAILED DESCRIPTION

In one aspect, the present invention provides a method for synthesizingTLR-4 receptor agonist E6020 having the structure:

E6020 is a potent TLR-4 receptor agonist, and thus the compound isuseful as an immunological adjuvant when co-administered with antigenssuch as vaccines for bacterial and viral diseases. For example, E6020may be used in combination with any suitable antigen or vaccinecomponent, e.g., an antigenic agent selected from the group consistingof antigens from pathogenic and non-pathogenic organisms, viruses, andfungi. As a further example, E6020 may be used in combination withproteins, peptides, antigens and vaccines which are pharmacologicallyactive for disease states and conditions such as smallpox, yellow fever,cancer, distemper, cholera, fowl pox, scarlet fever, diphtheria,tetanus, whooping cough, influenza, rabies, mumps, measles, foot andmouth disease, and poliomyelitis. In certain embodiments, E6020 and theantigen are each present in an amount effective to elicit an immuneresponse when administered to a host animal, embryo, or ovum vaccinatedtherewith.

In another aspect, the invention encompasses methods for synthesizingany stereoisomer of TLR-4 receptor agonist E6020. Thus there is providedherein a method for preparing a compound having the structure:

I. Preparation of Phosphoric Acid Ester Ureido Dimer

In certain embodiments, the inventive method comprises steps of:

(a) reacting a compound having the structure:

wherein R^(1a) is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl,heteroaryl, a phosphite oxygen protecting group, or a phosphate oxygenprotecting group; and

R^(2a) and R^(2b) are each independently hydrogen or a suitable nitrogenprotecting group, or R^(2a) and R^(2b), taken together, form a 5- or6-membered heterocyclic ring; wherein R^(2a) and R^(2b) are notsimultaneously hydrogen;

with phosgene under suitable conditions to effect formation of a ureidodimer having the structure:

(b) deprotecting ureido dimer (2) formed in step (a) under suitableconditions to effect formation of a partially deprotected dimer (3)having the structure:

(c) reacting the partially deprotected dimer formed in step (b) with asuitable reagent under suitable conditions to effect formation of aprotected dimer (4) having the structure:

and

(d) treating the dimer formed in step (c) with one or more suitablereagents under suitable conditions to effect formation of a sodium salthaving the structure:

In certain embodiments, compounds 1-5 above have the followingstereochemistry:

In yet other embodiments, the step of treating the dimer formed in step(c) with one or more suitable reagents under suitable conditions leadsto the formation of a compound having the structure:

which is then purified to yield the corresponding di-sodium salt:

In certain embodiments such as those shown above, each occurrence ofR^(1a) is independently hydrogen, a C₁-C₆ alkyl group, a C₃-C₆ alkenylgroup, a C₃-C₆ alkynyl group, or a phosphite oxygen protecting group orphosphate oxygen protecting group. In certain exemplary embodiments,each R^(1a) is allyl.

In certain embodiments, R^(2a) and R^(2b) are each independentlyhydrogen, alkyl, alkenyl, —C(═O)R^(x), —C(═O)OR^(x), —SR^(x), SO₂R^(x),or R^(2a) and R^(2b), taken together form a moiety having the structure═CR^(x)R^(y), wherein R^(2a) and R^(2b) are not simultaneously hydrogenand R^(x) and R^(y) are each independently hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl,heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl, heteroaryl,—C(═O)R^(A) or —ZR^(A), wherein Z is —O—, —S—, —NR^(B), wherein eachoccurrence of R^(A) and R^(B) is independently hydrogen, or an alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl,heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroarylmoiety. In certain exemplary embodiments, R^(2a) is hydrogen and R^(2b)is —C(═O)OR^(x), wherein R^(x) is substituted or unsubstituted loweralkyl. In certain other exemplary embodiments, R^(2a) is hydrogen andR^(2b) is —C(═O)OtBu.

In certain embodiments, the reaction conditions in step (a) comprisephosgene in a suitable solvent. In certain exemplary embodiments, thesolvent is CH₂Cl₂, toluene or combination thereof. In certainembodiments, the reaction conditions in step (a) additionally comprise aweak base. In certain exemplary embodiments, the weak base is aqueousNaHCO₃.

In certain embodiments, the deprotection reaction conditions in step (b)comprise a strong acid in a suitable solvent. In certain exemplaryembodiments, the solvent is CH₂Cl₂. In certain other exemplaryembodiments, R^(2a) is hydrogen, R^(2b) is —C(═O)OtBu and the strongacid is TFA.

In certain embodiments, the reagent of step (c) is a 3-oxo-tetradecanoicacid derivative. As used herein, “carboxylic acid derivative” (e.g.,3-oxo-tetradecanoic or dodecanoic acid derivative) refers to a compoundof structure RC(═O)X where R is the carboxyl radical and X is a chemicalgroup suitable to effect formation of an amide via reaction with aprimary amine, or that can be chemically transformed to effect formationof an amide via reaction with a primary amine. In certain embodiments, Xis halogen, hydroxyl, —OR, —SH, —SR or —C(halo)₃; where R is alkyl oraryl. In certain exemplary embodiments, the reagent is3-oxo-tetradecanoic acid. In certain embodiments, the reagent of step(c) is 3-oxo-tetradecanoic acid and the reaction conditions for reactingthe deprotected dimer with the reagent comprise a base. In certainembodiments, the base is 1-hydroxybenzotriazole. In certain embodiments,the base is Hunig's base. In certain embodiments, the reactionconditions of step (c) comprise a carboxylic acid activating reagentsuch as DCC. In certain embodiments, the carboxylic acid activatingreagent is 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide. In certainembodiments, the carboxylic acid activating reagent is HBTU.

In certain other embodiments, each occurrence of R^(1a) is allyl, andthe reaction conditions in step (d) comprise Pd(PPh₃)₄ in a suitablesolvent. In certain exemplary embodiments, the treating conditions instep (d) further comprise triphenyl phosphine and phenylsilane. Incertain exemplary embodiments, the solvent is THF.

In still other embodiments, purification of the compound having thestructure:

comprises chromatographic separation and treatment with a base. Incertain exemplary embodiments, the purification process comprises (i)ion exchange chromatography, (ii) C-4 Kromasil elution and (iii)treatment with aqueous NaOAc. In certain exemplary embodiments, thepurification process comprises (i) Biotage KP-silica chromatography,(ii) Biotage KP HS-C18 chromatography and (iii) treatment with aqueousNaOAc.

II. Method for Preparing Intermediate 1

In certain exemplary embodiments, the compound having the structure:

wherein R^(1a) is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl,heteroaryl or a phosphite oxygen protecting group or phosphate oxygenprotecting group; and

R^(2a) and R^(2b) are each independently hydrogen or a suitable nitrogenprotecting group, or R^(2a) and R^(2b), taken together, form a 5- or6-membered heterocyclic ring; wherein R^(2a) and R^(2b) are notsimultaneously hydrogen;

is prepared by a process comprising steps of:

(a) reacting an alcohol having the structure:

with a suitable partially protected diol having the structure:

wherein —OX¹ represents a suitable leaving group;

to form an alcohol having the structure:

(b) reacting alcohol 8 with a suitable dodecanoic acid derivative undersuitable conditions to form an ester having the structure:

(c) deprotecting ester 9 under suitable conditions to form a hydroxylamine having the structure:

(d) partially protecting hydroxyl amine 10 suitable conditions to forman alcohol having the structure:

wherein R^(2a) and R^(2b) are as defined above;

(e) treating alcohol 11 with one or more suitable reagents undersuitable conditions to effect formation of a phosphoric acid esterhaving the structure:

wherein R^(1a) is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl orheteroaryl, a phosphite oxygen protecting group or phosphate oxygenprotecting group; and

R^(3a) and R^(3b) are each independently hydrogen or a suitable nitrogenprotecting group, or R^(1a) and R^(3b), taken together, form a 5- or6-membered heterocyclic ring; wherein R^(3a) and R^(3b) are notsimultaneously hydrogen; and

(f) partially deprotecting 12 under suitable conditions to effectformation of amine 1:

In certain embodiments, each occurrence of R^(1a) is independentlyhydrogen, a C₁-C₆ alkyl group, a C₃-C₆ alkenyl group, a C₃-C₆ alkynylgroup, or a phosphite oxygen protecting group or phosphate oxygenprotecting group. In certain exemplary embodiments, each occurrence ofR^(1a) is allyl.

In certain embodiments, R^(2a) and R^(2b) are each independentlyhydrogen, alkyl, alkenyl, —C(═O)R^(x), —C(═O)OR^(x), SO₂R^(x), or R^(2a)and R^(2b), taken together form a moiety having the structure═CR^(x)R^(y), wherein R^(2a) and R^(2b) are not simultaneously hydrogenand R^(x) and R^(y) are each independently hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl,heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl, heteroaryl,—C(═O)R^(A) or —ZR^(A), wherein Z is —O—, —S—, —NR^(B), wherein eachoccurrence of R^(A) and R^(B) is independently hydrogen, or an alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl,heterocycloalkynyl, hetero aliphatic, heteroalicyclic, aryl orheteroaryl moiety. In certain exemplary embodiments, R^(2a) is hydrogenand R^(2b) is —C(═O)OR^(x), wherein R^(x) is substituted orunsubstituted lower alkyl. In certain other exemplary embodiments,R^(2a) is hydrogen and R^(2b) is —C(═O)OtBu.

In certain exemplary embodiments, X¹ is tosyl.

In certain embodiments, the dodecanoic acid derivative of step (b) isdodecanoic acid. In certain embodiments, the reagent of step (b) isdodecanoic acid and the reaction conditions for reacting alcohol 8comprise a base. In certain embodiments, the base is4-dimethylaminopyridine (DMAP). In certain embodiments, the reactionconditions of step (b) comprise a carboxylic acid activating reagentsuch as DCC. In certain embodiments, the carboxylic acid activatingreagent is 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide.

In certain embodiments, the deprotection reaction conditions of step (c)comprise catalytic hydrogenolysis and a suitable solvent. In certainexemplary embodiments, the deprotection reaction conditions of step (c)comprise H₂ and Pd/C. In certain exemplary embodiments, the solvent isisopropylalcohol (IPA).

In certain other exemplary embodiments, R^(2a) is hydrogen and R^(2b) is—C(═O)OtBu and the reaction conditions of step (d) comprisedi-tert-butyldicarbonate and a suitable solvent. In certain embodiments,the solvent is an alcohol. In certain exemplary embodiments, the solventis isopropylalcohol (IPA).

In certain embodiments, step (e) comprises:

(i) in situ formation of a phosphoramidous acid ester having thestructure:

wherein R^(4a) and R^(4b) are independently lower alkyl; and

(ii) in situ formation of a phosphorous acid ester having the structure:

wherein R^(1a), R^(2a), R^(2b), R^(3a) and R^(3b) are as defined above.

In certain other embodiments, the treating step (e) comprises aphosphorylating agent, and leads to the in situ formation ofphosphoramidous acid ester 13. In certain exemplary embodiments, thephosphorylating agent is allyl tetraisopropylphosphorodiamidite in thepresence of a dialkyl amine. In certain other embodiments, the treatingstep (e) comprises pyridinium trifuoroacetate. In certain exemplaryembodiments, the dialkyl amine is diidopropylamine and thephosphoramidous acid ester 13 has the structure:

In certain embodiments, the treating step (e) comprises in situformation of phosphoramidous acid ester 13, followed by reaction with aprotected ethanolamine having the structure:

wherein R^(3a) and R^(3b) are each independently hydrogen or a suitablenitrogen protecting group, or R^(3a) and R^(3b), taken together, form a5- or 6-membered heterocyclic ring; wherein R^(3a) and R^(3b) are notsimultaneously hydrogen. In certain exemplary embodiments, R^(3a) andR^(3b) are each independently hydrogen, alkyl, alkenyl, —C(═O)R^(x),—SR^(x), SO₂R^(x), or R^(3a) and R^(3b), taken together form a moietyhaving the structure ═CR^(x)R^(y), wherein R^(3a) and R^(3b) are notsimultaneously hydrogen and R^(x) and R^(y) are each independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl,heteroaliphatic, heteroalicyclic, aryl, heteroaryl, —C(═O)R^(A) or—ZR^(A), wherein Z is —O—, —S—, —NR^(B), wherein each occurrence ofR^(A) and R^(B) is independently hydrogen, or an alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl,heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroarylmoiety. In certain exemplary embodiments, R^(3a) is hydrogen and R^(3b)is —C(═O)OR^(x), wherein R^(x) is arylakyl. In certain other exemplaryembodiments, R^(3a) is hydrogen and R^(3b) is —C(═O)OR^(x), whereinR^(x) is 9-fluorenylmethyl (i.e., R^(3b) is Fmoc).

In certain exemplary embodiments, the treating step (e) comprises insitu reaction of phosphoramidous acid ester 13 with protectedethanolamine 15, where R^(3a) is hydrogen and R^(3b) is Fmoc, and thereaction conditions comprise acetic acid and pyridiniumtrifluoroacetate,

In certain embodiments, the treating step (e) comprises in situformation of phosphorous acid ester 14, followed by oxidation to formphosphoric acid ester 12:

in the presence of a suitable oxidizing agent. In certain exemplaryembodiments, the oxidizing agent is H₂O₂.

In certain embodiments, R²″ is hydrogen and R^(2b) is —C(═O)OtBu and thereaction conditions of step (f) comprise a dialkylamine and a suitablesolvent. In certain exemplary embodiments, the dialkylamine is dimethylamine. In certain other exemplary embodiments, the solvent is THF.

In certain embodiments, intermediates 6-13, 13a and 14 have thefollowing stereochemistry:

Synthetic Overview

The practitioner has a well-established literature of phospholipidchemistry to draw upon, in combination with the information containedherein, for guidance on synthetic strategies, protecting groups, andother materials and methods useful for the synthesis of E6020 andstereoisomers thereof.

The various patent documents and other references cited herein providehelpful background information on preparing certain monosaccharidestarting materials. In particular, certain reagents and startingmaterials are described in U.S. Pat. Nos. 6,551,600; 6,290,973 and6,521,776, the entirety of which are herein incorporated by reference.

Moreover, the practitioner is directed to the specific guidance andexamples provided in this document relating to exemplary intermediatesuseful for the synthesis of E6020 and stereoisomers thereof.

The compounds discussed above in the synthesis of E6020 have heptyl andundecyl side chains. These side chains may have varying lengths by usingappropriate reagents in the synthesis of E6020 analogs with differentalkyl chain lengths. Accordingly, the invention relates to compoundshaving the following formula (15):

In formula (15), A is —(CH₂)_(x)—O— or a covalent bond, n is 0 or 1, andx ranges from 1 to 6. When A is —(CH₂)_(x)—O—, the methylene group isbonded to the nitrogen atom in NR^(3a)R^(3b) and the oxygen is bound tothe phosphorous atom in the phosphite or phosphate group. Preferably xranges from 2 to 4 and most preferably 2. When n is 0, a compound offormula (15) contains a phosphite group. When n is 1, a compound offormula (15) contains a phosphate group.

R^(1a) is hydrogen, a C₁-C₆ alkyl group, a C₃-C₆ alkenyl group, a C₃-C₆alkynyl group, or a phosphite oxygen protecting group or phosphateoxygen protecting group. Such protecting groups are known in the art andan exemplary list is described above. A particularly preferred groupR^(1a) is an allyl group.

In formula (15), one of R^(2a) and R^(2b) is H and the other is amonovalent nitrogen protecting group; or R^(2a) and R^(2b) takentogether are a divalent nitrogen protecting group. For R^(3a) andR^(3b), when A is —(CH₂)—O—, one of R^(3a) and R^(3b) is H and the otheris a monovalent nitrogen protecting group or R^(3a) and R^(3b) takentogether are a divalent nitrogen protecting group. When A is a covalentbond, R^(3a) and R^(3b) are independently selected from C₁-C₆ alkyl ortaken together are —(CH₂)₄—, —(CH₂)₅—, or —(CH₂)₂O(CH₂)₂—. Preferably,when A is a covalent bond, R^(3a) and R^(3b) are C₂-C₆ alkyl groups suchas ethyl, propyl or butyl and more preferably isopropyl groups.

In compounds of formula (15), the protecting group on the nitrogenlinked to R^(2a) and R^(2b) can be removed under a first conditionselected from acidic, basic, oxidative, and reductive conditions; andthe protecting group on the nitrogen linked to R^(3a) and R^(3b) can beremoved under a second condition selected from the remaining threeconditions that are different from the first condition. Preferrednitrogen protecting group selected from the group consisting of Boc,Fmoc, TROC, TMS-ethylcarbonyl, cyanoethylcarbonyl, allyloxycarbonyl,(C₆H₅)₂C═, tetrachlorophthalamide, or azide. Generally speaking, thereare four types of conditions which may be used to remove nitrogenprotecting groups, acidic, basic, oxidation or reductive conditions. Ina preferred embodiment, one nitrogen protecting group is selectivelyremoved under one of these four conditions and the other nitrogenprotecting group is selectively removed using one of the remaining threeconditions. In one embodiment the nitrogen protecting groups arerespectively removed with mild acidic or mild basic conditions.

The nitrogen linked to R^(2a) and R^(2b) could be protected with a Bocgroup and the nitrogen linked to R^(3a) and R^(3b) could be protectedwith an Fmoc group or vice versa. The Boc group can be selectivelyremoved under acidic conditions (methanesulfonic acid, trifluoroaceticacid, or formic acid in a solvent such as methylene chloride at roomtemperature). The Fmoc group could be selectively removed while usingsecondary amines like piperidine or dimethylamine in a solvent such asTHF at room temperature.

Alternatively, the nitrogen linked to R^(2a) and R^(2b) could beprotected with a Troc group and the nitrogen linked to with R^(3a) andR^(3b) could be protected with an Fmoc group or vice versa. The Fmocgroup could be selectively removed under conditions described above andthe Troc group could be cleaved under reducing conditions such as zincin THF, water.

In another example, the nitrogen linked to R^(2a) and R^(2b) could beprotected with a Troc group and the nitrogen linked to R^(3a) and R^(3b)could be protected with a Boc group or vice versa. The Troc group couldbe cleaved under reducing conditions such as zinc in THF, water and theBoc group could be selectively removed under conditions as describedabove.

R⁴ is a C₅-C₁₂ alkyl group or a C₅-C₁₂ alkenyl group. R⁴ is a C₅-C₁₂alkyl group; preferably a C₅-C₉ alkyl group, more preferably a C₇ alkylgroup, and most preferably an n-heptyl group.

R⁵ is a C₅-C₁₅ alkyl group or a C₅-C₁₅ alkenyl group. R⁵ is a C₅-C₁₅alkyl group, preferably a C₇ to C₁₃, more preferably a C₁₁ alkyl group,and most preferably, n-undecyl.

Salts of the compounds of formula (15) may occur during synthesis or mayalso be made by reacting a compound of formula (I) with an acid or abase. Acid addition salts are preferred.

Preferred compounds of formula (15) are those (a) wherein A is—(CH₂)₂—O—; n is 0; R⁴ is a C₇ alkyl; and R⁵ is a C₁₁ alkyl; (b) whereinA is —(CH₂)₂—O—; n is 1; R⁴ is a C₇ alkyl; and R⁵ is a C₁₁ alkyl; (c)wherein A is a covalent bond, n is 0; R⁴ is a C₇ alkyl; and R⁵ is a C₁₁alkyl; and (d) wherein A is a covalent bond, n is 0; R^(3a) and R^(3b)are each isopropyl; R⁴ is a C₇ alkyl; and R⁵ is a C₁₁ alkyl.

Such intermediates are useful in preparing E6020 analogs and precursorshaving the following formula (16):

In formula (16), n is 0 or 1 as discussed above for formula (I). R^(1a)is hydrogen, a C₁-C₆ alkyl group, a C₂-C₆ alkenyl group, a C₂-C₆ alkynylgroup, a phosphite oxygen protecting group, or a phosphate oxygenprotecting group. Preferred substituents for group R^(1a) are the sameas those discussed above. For example, one of R^(2a) and R^(2c) is H andthe other is a monovalent nitrogen protecting group or —C(O)CH₂C(O)R⁶;or R^(2a) and R^(2c) taken together are a divalent nitrogen protectinggroup. Preferable groups for R^(2a) and R^(2c) are the same as thosedescribed above for R^(2a) and R^(2b), except that one of R^(2a) orR^(2c) may also preferably be —C(O)CH₂C(O)R⁶. R⁴ is a C₅-C₁₂ alkyl groupor a C₅-C₁₂ alkenyl group with the same preferred groups as in formula(15). R⁵ and R⁶ are independently a C₅-C₁₅ alkyl group or a C₅-C₁₅alkenyl group with the preferred substituents being the same as thosedescribed above for R⁵.

Preferred compounds of formula (16) are those (a) wherein n is 1, R⁴ isa C₇ alkyl, and R⁵ is a C₁₁ alkyl, wherein n is 1, R^(1a) is allyl,R^(2a) is hydrogen, R^(2c) is Boc, R⁴ is a C₇ alkyl, and R⁷ is a C₁₁alkyl; (c) wherein n is 1, R^(2a) is hydrogen, R^(2a) is —C(O)CH₂C(O)R⁶,R⁴ is a C₇ alkyl, R⁵ is a C₁₁ alkyl, and R⁶ is a C₁₁ alkyl; (d) whereinn is 0, R^(1a) is allyl, R^(2a) is hydrogen, R^(2c) is Boc, R⁴ is a C₇alkyl, and R⁵ is a C₁₁ alkyl; (e) wherein n is 1, R^(1a) is allyl,R^(2a) is hydrogen, R^(2c) is hydrogen, R⁴ is a C₇ alkyl, and R⁵ is aC₁₁ alkyl; and (f) wherein n is 0, R^(1a) is allyl, R^(2a) is hydrogen,R^(2c) is —C(O)CH₂C(O)R⁶, R⁴ is a C₇ alkyl, R⁵ is a C₁₁ alkyl, and R⁶ isa C₁₁ alkyl.

Salts of the compounds of formula (16) may occur during synthesis or mayalso be made by reacting a compound of formula (I) with an acid or abase. Acid addition salts are preferred.

The invention also includes compounds of formula (17):

In formula (17), R is hydrogen or a C₁-C₆ alkyl group and preferablyhydrogen. One R^(2a) and R^(2b) in formula (17) is H and the other is amonovalent nitrogen protecting group; or R^(2a) and R^(2b) takentogether are a divalent nitrogen protecting group. The preferred groupsfor R^(2a) and R^(2b) are those discussed above for formula (15). R⁴ isa C₅-C₁₂ alkyl group or a C₅-C₁₂ alkenyl group and R⁵ is a C₅-C₁₅ alkylgroup or a C₅-C₁₅ alkenyl group. The preferred groups for R⁴ and R⁵ arealso those discussed above for formula (15).

Preferred compounds of formula (17) include those (a) wherein R ishydrogen, R^(2a) and R^(2b) are hydrogen, R⁴ is a C₇ alkyl, and R⁵ is aC₁₁ alkyl; and (b) wherein R is hydrogen, R^(2a) is hydrogen, R^(2b) isa nitrogen protecting group, R⁴ is a C₇ alkyl, and R⁵ is a C₁₁ alkyl.

The invention also includes compounds of formula (18):

In formula (18), R⁴ is a C₅-C₁₂ alkyl group or a C₅-C₁₂ alkenyl group;and R⁵ is a C₅-C₁₅ alkyl group or a C₅-C₁₅ alkenyl group. Preferredgroups for R⁶ and R⁷ are those discussed above. A preferred compound offormula (18) is ER-819509.

Examples of synthetic methods for practicing the invention are providedbelow, as detailed in Schemes 1-5, and in the Exemplification herein. Itwill be appreciated that the methods as described herein can be appliedto each of the compounds as disclosed herein and equivalents thereof.Additionally, the reagents and starting materials are well known tothose skilled in the art. Although the following schemes describecertain exemplary intermediates and protecting groups, it will beappreciated that the use of alternate starting materials, protectinggroups and/or reagents will yield other intermediates, which areconsidered to fall within the scope of the present invention.

In certain exemplary embodiments of the present invention, preparationof the ester ER-819059 is achieved in three steps selectivelyfunctionalizing diol ER-028694 with a suitable group, therebytransforming the primary hydroxyl into a leaving group. For example, theprimary hydroxyl of diol ER-028694 is tosylated to give thecorresponding adduct ER-028695. Tosyl ester ER-028695 is then coupledwith alcohol ER-807277 in the presence of a base, such as NaHMDS to givethe corresponding ether ER-806158. Esterification of alcohol ER-806158with lauric acid, followed by hydrogenolysis of the phenyldihydrooxazole group leads to hydroxylamine ER-819120. Boc protection ofthe amino group gives ER-819302.

Boc-protected ER-819302 may then be converted to E6020 in 6 steps, asoutlined in Schemes 2 and 3. For example, phosphorylation of ER-819302in the presence of bis(allyloxy)diisopropyl aminophosphine anddiisopropylamine, followed by reaction in situ with FmocNH(CH₂)₂OH, andoxidation (e.g., hydrogen peroxide) leads to the formation ofphosphorylated intermediate ER-819344, which, upon deprotection insuitable conditions (e.g., Me₂NH) leads to the formation of deprotectedintermediate ER-819385. Reaction of ER-819385 with phosgene in suitableconditions (e.g., 20% phosgene in toluene in the presence of aqueousNaHCO₃) leads to the formation of diphosphoric acid ester ER-819409.Deprotection of the Boc-protected secondary amines on ER-819409 using anappropriate acid such as TFA provides the diamine intermediateER-807284. Amidation of the free amines using 3-oxo-1-tetradecanoic acidin the presence of a coupling reagent such as EDC and DMAP provides thepenultimate product ER-807285. Deprotection of the allyl-phosphateesters using a palladium (0) reagent such as palladium (0)tetrakistriphenylphosphine in the presence of excess triphenylphosphineand a proton source (phenylsilane) provides the desired, crude productthat can be purified (e.g., suitable ion exchange chromatographicconditions, followed by treatment with aqueous NaOAc) to give thedesired compound E6020.

It will be appreciated that each of the reactions described in Schemes1, 2 and 3 above can be carried out using reagents and conditions asdescribed for the synthesis of various types of exemplary intermediatesdescribed above, or they may be modified using other available reagents,protecting groups or starting materials. For example, a variety of ureaformation conditions, phosphorylation and amine protecting/deprotectingconditions are well-known in the art and can be utilized in the methodof the invention. See, generally, March, Advanced Organic Chemistry,5^(th) ed., John Wiley & Sons, 2001; and “Comprehensive OrganicTransformations, a guide to functional group preparations”, Richard C.Larock, VCH publishers, 1999; the entire contents of which areincorporated herein by reference and “Protective Groups in OrganicSynthesis” Third Ed. Greene, T. W, and Wuts, P. G., Eds., John Wiley &Sons, New York: 1999, the entire contents of which are herebyincorporated by reference.

In summary, the present invention provides a synthesis of E6020 insignificantly fewer and higher yielding steps than previously reportedmethods. The instant method affords the title compound in high overallyields and through a highly efficient route.

The representative examples that follow are intended to help illustratethe invention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the exampleswhich follow and the references to the scientific and patent literaturecited herein. It should further be appreciated that the contents ofthose cited references are incorporated herein by reference to helpillustrate the state of the art.

The following examples contain important additional information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and the equivalents thereof.

EXEMPLIFICATION

The compounds of this invention and their preparation can be understoodfurther by the examples that illustrate some of the processes by whichthese compounds are prepared or used. It will be appreciated, however,that these examples do not limit the invention. Variations of theinvention are considered to fall within the scope of the presentinvention as described herein and as hereinafter claimed.

The compounds of this invention and their preparation can be understoodfurther by the examples that illustrate some of the processes by whichthese compounds are prepared or used. It will be appreciated, however,that these examples do not limit the invention. Variations of theinvention, now known or further developed, are considered to fall withinthe scope of the present invention as described herein and ashereinafter claimed.

Using the preparation of immunological adjuvant E6020 to exemplify, thefollowing Examples encompass the synthesis of synthetic precursors ofimmunological adjuvant compounds using the methods and compounds of thepresent invention.

One of ordinary skill in the art would recognize that many analogs ofE6020 are prepared by the methods and from the compounds of the presentinvention including, but not limited to, those analogs of E6020described in U.S. Pat. Nos. 6,551,600; 6,290,973 and 6,521,776, theentirety of which are herein incorporated by reference. Accordingly, itwill be appreciated that the synthetic methods described below, by wayof example, do not limit the scope of the invention which is defined bythe appended claims.

General Reaction Procedures

Unless mentioned specifically, reaction mixtures were stirred using amagnetically driven stirrer bar. An inert atmosphere refers to eitherdry argon or dry nitrogen. Reactions were monitored either by thin layerchromatography, by proton nuclear magnetic resonance or by high-pressureliquid chromatography (HPLC), of a suitably worked up sample of thereaction mixture.

Listed below are abbreviations used for some common organic reagentsreferred to herein:

-   -   ATP: Allyl tetraisopropylphosphorodiamidite    -   DMF: Dimethyl formamide    -   EA: Ethyl Acetate    -   EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride    -   HBTU:        O-Benzotriazole-N,N,N′,N′-tetramethyluronium-hexafluoro-phosphate    -   HOBT: 1-Hydroxybenzotriazole    -   IPA: Iso-propyl alcohol    -   MTBE: Methyl tert-butyl ether    -   NaHMDS: Sodium hexamethyldisilazane    -   Pyr.TFA: Pyridinium trifluoroacetate    -   TBME: Tert-butyl methyl ether    -   TFA: Trifluoro acetic acid    -   THF: Tetrahydrofuran    -   TsCl: Tosyl chloride

Example 1 Preparation of ER-028695

Preparation 1:

To a stirred solution of p-toluenesulfonyl chloride (1704 g, 8.938 mol)in dry tetrahydrofuran (THF) (2408 g) in a dry 22-L reactor under anitrogen atmosphere at 0° C. was added ER-028694 (952 g, 5.46 mol) indry THF (1041 g) over a 9-minute period. The residual ER-028694 wasrinsed with anhydrous THF (364 g) into the reactor. Triethylamine (1.35Kg, 13.4 mol) was added drop wise to the clear yellow reaction solutionover a 19-minute period during which time the solution turned cloudy.The residual triethylamine was rinsed with dry THF (30 g) into thereaction mixture.

After stirring for an additional 15 hours 17 minutes at 0° C., thereaction was quenched with a slow addition of 1.0 M of hydrochloric acid(2120 g) over a 36-minute period with a temperature change from 0.1 to5.9° C. Brine (785 mL) was added over a 3-minute period, stirringcontinued for an additional 5 minutes followed by transferring thereaction mixture (˜22 L) to a 50-L work-up reactor with THF (0.57 Kg).The organic layer was separated from the aqueous layer (pH=6). Theorganic layer was washed with a mixture of 1.0 M hydrochloric acid (2120g) and brine (785 mL). The organic layer was then washed with a mixtureof water (2120 mL) and brine (980 mL).

The organic layer was transferred to a clean 22-L reactor followed bythe addition of THF (1.6 Kg) under a nitrogen atmosphere. The solutionwas cooled to 0° C. (ice-water bath) with stirring followed by theaddition of 10% aqueous NaOH solution (2.04 Kg) over a 7-minute periodwith a temperature change from −0.4 to 2.3° C. After the addition of 28%aqueous NH₄OH solution (935 g) over a 6-minute period with a temperaturechange from 1.9 to 12.5° C., the mixture was stirred for 25 minutesfollowed by the addition of heptane (1.1 Kg). The reaction mixture wastransferred to a 50-L work-up reactor with heptane (0.532 Kg), mixedwell, allowed to settle, and the aqueous layer (pH 14) was discarded.The organic layer was washed with 10% aqueous NaOH (2.04 Kg) followed bywater (2.04 Kg). Evaporation of the organic layer solvent (house vacuum)and azeotroping the residue with heptane (1.1 Kg) provided a clear,orange oil. The isolated material (1.839 Kg) was used in the next stepwithout further purification. Analytical Data for ER-028695: ¹H-NMR(CDCl₃, 400 MHz) δ 0.88 (t, J=7.1 Hz, 3H), 1.2-1.5 (m, 12H), 1.57 (bs,1H), 1.6-1.7 (m, 1H), 1.8-1.9 (m, 1H), 2.45 (s, 3H), 3.7-3.8 (m, 1H),4.1-4.2 (m, 1H), 4.2-4.3 (m, 1H), 7.35, (d, J=7.8 Hz, 2H), 7.80 (d,J=7.8 Hz, 2H). MS-APESI (M+Na) Calcd for C₁₇H₂₈NaO₄S: 351.16 Found:351.23. HPLC: ER-028695:ER-817864 ratio of 87.11%:11.71%.

Preparation 2:

Anhydrous THF (250 mL) was added to a stirred solution ofp-toluenesulfonyl chloride (161.1 g, 0.845 mol) under a nitrogenatmosphere. ER-028694 (90.0 g, 0.516 mol) was then added followed bytriethylamine (176 mL, 1.26 mol) at a reactor temperature of 22° C. togive clear yellow solution. The solution turned cloudy after a fewminutes. After stirring for 15 h 33 min, a 100-4 sample was taken. GCanalysis showed 100% conversion. Another 100-μL sample was taken, andextracted with 2.0 mL process water and 3.0 mL heptane. The organiclayer was washed with process water (2.0 mL) twice, and then with brine(2.0 mL). The resulting organic layer was evaporated under reducedpressure to give a colorless oil. ¹H-NMR analysis of the colorless oilshowed an ER-028694/ER-028695/ER-817864 ratio of 1.00/91.93/7.07.

After the reaction was determined to be complete, 1.0 M of hydrochloricacid solution was added with a temperature change from 21.4° C. to 26.8°C., followed by an addition of brine (74.2 mL). The organic layer (˜0.6L) was separated from the aqueous layer (˜0.4 L, pH=10) and washed witha mixture of 1.0 M hydrochloric acid (201 mL) and brine (74.2 mL)followed by a mixture of process water (201 mL) and brine (92.7 mL). Theorganic layer was then transfer to a clean reactor followed by theaddition of THF (200 mL) under a nitrogen atmosphere. The solution wascooled to 10° C. with stirring followed by the addition of 10% aqueousNaOH solution (193 g) over 2 min while keeping the reaction temperaturebelow 25° C. (temperature changed from 15.3° C. to 21.3° C.). An aqueousNH₄OH solution (88.4 g) was then added over a 5-min period (delayedexotherm was observed) while still keeping the reaction temperaturebelow 25° C. (temperature changed from 15.5° C. to 22.7° C.). Thereaction mixture was warmed to 20° C. under stirring for 15 min. Thereaction mixture was extracted from the aqueous layer with heptane (200mL), with the aqueous layer having a pH of 14. The organic layer waswashed with 10% aqueous NaOH (193 g) followed with process water (193mL). Evaporating the solvent (at 29-32° C., 20 torr) and chasing theresidue with heptane (200 mL, 29-32° C., 4.4 torr) produced a clear,orange oil. The isolated material was used in the next step (assuming100% yield) and without further purification. ¹H-NMR analysis of thecolorless oil gave an ER-028694/ER-028695/ER-817864 ratio of0.9/90.9/8.2, KF was 13.0 ppm.

Example 2 Preparation of ER-806158

Preparation 1:

To a stirred solution of ER-807277 (1.177 Kg, 6.642 mol) in dry THF(10.580 Kg) in a dry 22-L reactor under a nitrogen atmosphere at −1° C.was added 1.0 M sodium bis(trimethyldisilyl)amide in THF (3.300 Kg) overa 33-minute period while keeping the reaction temperature between−0.8-4.1° C. After stirring the solution for an additional 17 minutes at−0.1° C., crude ER-028695 (1.089 Kg, 3.316 mol) in dry THF (0.968 Kg)was added to the reaction solution over a 5-minute period maintainingthe temperature between −0.1-3.9° C. The residual ER-028695 was rinsedinto the reactor with dry THF (0.253 Kg), The final reaction mixture waswarmed to room temperature during which time the orange clear reactionsolution turned into a suspension (approx. at 19° C.). After stirringfor 3 hours 31 minutes at room temperature, the completed reactionmixture was cooled to 0.1° C. and poured into a mixture of coldsaturated aqueous NH₄Cl (4.7 Kg) and brine (1.65 L) in a 50-L work-upreactor (exothermic, Tmax=16.2° C.). Water (2×1.0 L) followed by toluene(3.769 Kg) was used to rinse the residual reaction mixture to thework-up reactor. After stirring the mixture for 5 minutes, the reactionmixture was allowed to settle for 5 minutes and the resultant bottomaqueous layer (pH=9) was discarded. The solvent of the organic layer waspartially evaporated under house vacuum at 30-34° C. The container wasrinsed with minimum THF (0.4 Kg) and the rinse was combined with theproduct. Toluene (0.4 Kg) was added with heating (bathtemperature=30-34° C.) to break up the solid into a slurry for ease oftransfer.

The crude slurry was transferred to a clean, dry 22-L reactor undernitrogen followed by heptane (7.503 Kg) with fast stirring at ˜19° C.After additional stirring for 6 hours 22 minutes, the mixture wassubjected to vacuum filtration (Fisher P5 filter paper, catalog#09-801G), and the cake (˜5.4 L) washed with heptane three times: 0.99Kg, 1.02 Kg and 1.01 Kg, respectively. The combined filtrate and washeswere evaporated at 30-34° C. under house vacuum to give a cloudy orangeoil (1.207 Kg). The white solid cake in the filter funnel is ER-807277.

The crude product (1.206 Kg) was dissolved in methyl tert-butyl ether(MTBE)/heptane=1/4 (700 mL), filtered through a medium fritted filterfunnel followed by rinsing the filter cake with MTBE/heptane=1/4 (300mL) to give a clear yellow filtrate. The crude ER-806158 solution wasloaded onto a pre-conditioned silica gel cartridge [Biotage 150L (5.62Kg, void volume=7.07 L) cartridge conditioned with MTBE (10.46 Kg)followed by MTBE/heptane=1/4 (3.140 Kg/11.606 Kg)] using an adjusted theflow rate to ˜840 mL/min (˜25 psi solvent pressure). After loading, thecartridge was rinsed with MTBE/heptane=1/4 (0.148 Kg/0.547 Kg) followedby elution with MTBE/heptane=1/4 (2.09 Kg/7.740 Kg), withMTBE/heptane=2/3 (2.094 Kg/2.904 Kg, with MTBE/heptane=7/3 (3.661Kg/1.448 Kg), and finally with MTBE (40.298 Kg). Approximately 350-mLfractions were collected during the entire chromatography process. Thecombined, product containing fractions were concentrated and azeotropedto dry using heptane (0.540 Kg) followed by drying under house vacuum at33° C. for 1 hour 13 minutes to give a clear orange oil (0.793 Kg, 71%)at 98.32 area % purity.

Crystallization of ER-806158

2.376 Kg of purified ER-806158 was dissolved in heptane (8.121 Kg) andtransferred to a clean dry 22-L reactor under a nitrogen atmospherefollowed by stirring. The clear yellow solution was cooled to −15° C.stepwise at ˜4° C. every 0.5 h. The resulting white suspension wasstirred for an additional 1 hour at −15° C. followed by filtration overa chilled filter funnel and filter paper using vacuum with a nitrogenblanket over the filter cake. The desired solid was washed with coldheptane (−12.3° C., 1.387 Kg) filtered as above and the filter cake waskept under vacuum for 14 hours 13 minutes (T=14.9-18.8° C.) whileapplying a nitrogen blanket on top of the solid. The mother liquor,washes, and the solvent to dissolve residual ER-806158 in the reactorwere combined for recycles.

Obtained: 2.068 Kg, 87.0% yield

Analytical Data for ER-806158

Purity: 99.84 wt/wt %

Chiral purity: 99.72% e.e.

KF: 0.21%

Heptane: 268 ppm

¹H-NMR (CDCl₃) δ 0.88 (t, J=6, 9 Hz, 3H), 1.2-1.6 (m, 12H), 1.6-1.7 (m,1H), 1.7-1.8 (m, 1H), 3.21 (bs, 1H), 3.6-3.7 (m, 3H), 3.7-3.8 (m, 2H),3.5-3.6 (m, 2H), 4.2-4.3 (m, 1H), 4.5 (m, 2H), 7.42, (t, J=7.6 Hz, 2H),7.49, (dd, J=6, 9, 7.8 Hz, 1H), 7.95 (d, J=7.3 Hz, 2H).

Elemental Analysis (EA): Calcd for C₃₀H₅₉NO₆: C, 72.04; H, 9.37; N; 4.20Found: C, 71.83; H, 9.30; N, 4.08.

m.p. (DSC) Onset, 26.7° C.; Maximum, 29.5° C.

Preparation 2:

The quantity (mol) of ER-028695 used in the reaction was calculatedbased on the amount of starting ER-028694 used in Example 1, procedure2, assuming 100% yield. ER-807277 (178.3 g, 1006 mmol) was added to aclean dry 5-L reactor under a nitrogen atmosphere. Dry THF (1.8 L) wasadded with stirring to produce a clear, yellow solution, and thesolution was cooled to 0° C. NaHMDS/THF (1.0 M, 553 mL) was added over a20 minute-period and the reaction temperature was kept at less than orequal to 5° C. (3.8-1.3° C.). The solution was stirred for 10 minutesand then cooled to −5° C. Crude ER-028694 (calculated to be 165 g, 503mmol) was transferred with dry THF (165 mL, 1 vol.) to a dry clean flaskunder a nitrogen atmosphere with stirring, which produced a clear,orange solution. The ER-028695/THF solution was then added to thereaction solution over a 6-minute period, with the temperature changingfrom −3.2° C. to 0.4° C. Residual ER-028695 was rinsed into the reactorwith dry THF (40 mL) and the reaction mixture was warmed to roomtemperature in a warm water bath (23° C.). During the warm-up process,the clear, orange reaction solution turned into a suspension. After 1.75h of stirring at 18.7-21.3° C., a sample (10 μL) was taken, added to 1.0mL MeCN, and filtered through 0.2 μm syringe filter to give a colorlessclear filtrate, HPLC analysis (TM-779) detected 98.2% conversion. After3 h total stirring at room temperature, HPLC analysis showed 99.5%conversion. Saturated aqueous NH₄Cl (0.66 L) was added in one portion(exothermic, 18.9-26.0° C.), followed by additions of process water(0.30 L), brine (0.25 L), and toluene (0.66 L), Stirring was continuedfor 5 minutes after the additions. The solvent of the top organic layer(˜2.6 L) was partially evaporated under reduced pressure at 29-32° C. togive a slurry (481.7 g), and the bottom aqueous layer (˜1.4 L, pH-11)was discarded.

The slurry was transferred to a dry clean 5-L reactor under nitrogen.Heptane (1.65 L) was added at room temperature with fast stirring thatcontinued for 14 h. The mixture was subjected to vacuum filtration(medium filter paper), the resulting cake (˜0.52 L) was washed withheptane (0.33 L), and the yellow filtrate (˜2.8 L) was collected.Solvent evaporation (29-32° C.) under reduced pressure produced anorange oil (177.9 g).

The crude product (177.9 g) was dissolved in TBME/heptane=1/4 (178 mL)and loaded onto a Biotage 75L silica gel (834 g, void volume=1.05 L)cartridge conditioned with TBME (2.1 L) followed by TBME/heptane=1/4(3.15 L) using an adjusted flow rate of −170 mL/min (˜18 psi solventpressure). Fractions 1-2 (˜350 mL each fraction) were collected. Afterloading, the cartridge was rinsed with TBME/heptane=1/4 (178 mL) andeluted with TBME/heptane=1/4 (2.1 L), collecting fractions 2-8; elutedwith TBME/heptane=2/3 (1.05 L), collecting fractions 8-11; eluted withTBME/heptane=7/3 (1.05 L), collecting fractions 11-14; and eluted withTBME (8.4 L), collecting fractions 15-39. The collected fractions wereanalyzed (TLC, silica gel F254; mobile phase, TBME; visualization, UVand anisaldehyde solution), and the product containing fractions (14-35)without ER-807277 contamination were combined. Solvent evaporation(29-32° C., 80 torr) chased with heptane (29-32° C., 10.3 torr) produceda clear, orange oil (114.4 g), which solidified upon cooling to roomtemperature. HPLC analysis (TM-779) detected 96 area %.

The orange solid (114.4 g, 1 wt) was warmed (29-32° C.) and transferredwith heptane (572 mL) to a 1-L dry clean reactor under a nitrogenatmosphere with stirring. The solution was cooled 14° C., stirred for0.5 h, and seeded with ER-806158 crystals (0.112 g). Stirring wascontinued for 0.5 h, at which time solid particles were visible. Thereaction mixture was cooled stepwise to −10° C. (4° C. every 0.5 h),further cooled to −15° C., while stirring continued for 1 h. A chilled,medium filter funnel was prepared with cold (˜−20° C.) heptane (˜200mL), and the reaction mixture was filtered through the funnel by vacuum,followed by a wash with cold heptane (˜−20-−15° C.). The vacuum wascontinued for 1.5 h and a nitrogen blanket was applied on top of thecake. The resulting white coarse, granular solid was then transferred toa bottle (98.4 g, 0.295 mol). Analytical results showed: wt/wt %, 99.95;purity, 99.68; residual heptane, 19.1 ppm; KF, 1.04%. DSC showed melton-set was 27° C., Chiral HPLC analysis (TM-573) detected 99.8 area %.

Example 3 Preparation of ER-819059

Preparation 1:

To a stirred solution of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (904 g, 4.176 mol), lauric acid (purified, 881 g, 4.398mol), and ER-806158 (1400 g, 4.198 mol) in dry dichloromethane (CH₂Cl₂)(3710 g) in a clean dry 22-L flask under a nitrogen atmosphere was added4-dimethylaminopyridine (51 g, 0.417 mol). After stirring for 1 hour 13minutes, the reaction mixture turned into a slightly cloudy yellowsolution and the reaction temperature reached a maximum of 26.7° C.After 6 h 34 minutes the reaction was found to be 98.9% complete, atwhich time additional N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (303 g, 1.581 mol) was added in one portion. Afterstirring for a total of 20 hours 50 minutes at room temperature, MeOH(5566 g) was added in one portion to the yellow reaction suspension(slightly endothermic, T_(min)=17.1° C.). Partial solvent evaporation(house vacuum, 29-32° C.) to remove the CH₂Cl₂ (3.71 Kg) was followed byextraction with equal portions of heptane (2×2.86 kg ea.) The heptaneextracts were evaporated (house vacuum, 30-35° C.) and azeotroped todryness using heptane (0.75 Kg).

The product (2.192 Kg) was dissolved in heptane (3.00 Kg) and loadedonto a silica gel cartridge [Biotage 150L silica gel (5.62 Kg)pre-conditioned with MTBE/heptane=1/1 (15.0 Kg)]. The product was elutedat an adjusted flow rate of ˜0.84 L/min (solvent pressure=22 psi) withMTBE/heptane=1/1 (31.3 Kg.) collecting approx. 350 mL eluant/fraction.The product containing fractions were combined and concentrated todryness and vacuum dried (16° C., house vacuum, 16 hours 13 minutes) togive ER-819059 (2.102 Kg, 86.4% yield) as a clear, pale yellow oil.

Analytical Data for ER-819059

HPLC analysis: 99.76 area %.

KF: 0.36%

Heptane: 725 ppm

¹H-NMR (CDCl₃) δ 0.881 (t, J=7.1 Hz, 3H), 0.884 (t, J=6.9 Hz, 3H),1.2-1.3 (m, 26H), 1.5-1.6 (m, 2H), 1.6-1.7 (m, 2H), 1.8-1.9 (m, 2H),2.28 (t, J=7.6 Hz, 2H), 3.4-3.6 (m, 3H), 3.69 (dd, J=3.7, 9.6 Hz, 1H),4.3-4.6 (m, 3H), 5.0 (m, 1H), 7.43, (t, J=7.6 Hz, 2H), 7.51, (dd, J=6.0,7.3 Hz, 1H), 7.95 (d, J=6.0 Hz, 2H).

MS-APESI (M+H) Calcd for C₃₂H₅₄NO₄: 516.41 Found: 516.48

TLC: (silica gel F254): MTBE/heptane=1/1; R_(f) of ER-819059=0.51

Preparation 2:

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.49 g,7.77 mmol), ER-806158 (2.00 g, 6.00 mmol), DMAP (0.07 g, 0.59 mmol) anddodecanoic acid (1.44 g, 7.19 mmol) were added to a dry 50-mL flaskunder a nitrogen atmosphere. Dry dichloromethane (6.0 mL) was then addedwith stirring. The stirring was continued at room temperature until aslightly cloudy solution resulted. After stirring for 16 h, a sample (10μL) was taken and added to 1.5 mL MeCN. HPLC analysis (TM-780) detected95.1% conversion. An additional amount ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (0.307 g,1.60 mmol) was then added. After another 8 h of stirring (24 h total), asecond sample (10 μL) was taken, and analyzed as before. HPLC analysis(TM-780) detected 99.6% conversion. The stirring was continued foranother 15 h (39 h total), at which time a third sample was taken andanalyzed. HPLC (TM-780) analysis detected 99.96% conversion. Saturatedaqueous NaHCO₃ (10 mL), brine (10 mL), and heptane (10 mL) were thenadded while stirring continued for 0.5 h. A poor emulsion was observed.Heptane (10 mL) was then added, and mixed well, but did notsubstantially improve the emulsion, Next, MeOH (3.0 mL) was added, andmixed well, but it also did not substantially improve the emulsion. Thecomposition was allowed to settle for 0.5 h, and the milky aqueous layerthat formed on the bottom was drained. The aqueous layer was extractedwith TBME (20 mL), and the bottom aqueous layer was drained after it wasallowed to settle for about 15 min. TLC analysis (TLC, silica gel F254;mobile phase, TBME; visualization, UV and anisaldehyde solution) of theaqueous layer detected a significant amount of product. The aqueouslayer was again extracted with TBME (20 mL) and the organic layers werecombined. Solvent evaporation (29-32° C., 7.5 torr) producted a clear,yellow oil (3.35 g).

The crude product (3.35 g) was dissolved in TBME/heptane=1/1 (12 mL) andloaded onto Biotage 12M silica gel (8.99 g, void volume=11.3 mL)cartridge conditioned with TBME/heptane=1/1 (36 mL). The product waseluted at an adjusted flow rate of ˜12 mL/min, collecting 15 fractions(˜6 mL each). The collected fractions were analyzed (TLC, silica gelF254; mobile phase, TBME/heptane=1/1; visualization, UV and anisaldehydesolution), and fractions containing product (3-15) were combined.Solvent evaporation (29-32° C., 8.3 torr) produced a clear, colorlessoil (3.03 g). HPLC analysis (TM-780) detected 99.18 area %, The aqueousworkup was not performed because of the poor emulsion.

Example 4 Preparation of ER-819120

ER-819059 (3.03 g, 5.87 mmol, 1 wt) and 10% Pd/C (0.303 g, 0.28 mmol)were added to a clean 50-mL flask with IPA (20.2 mL, 6.67 vol.) under anitrogen atmosphere. The flask was evacuated with fast stirring at roomtemperature and purged with hydrogen (hydrogen pressure maintained at0.04 bar). This evacuation-and-purging process was repeated twoadditional times. The reaction was monitored through HPLC analysis ofvarious samples After the reaction was complete, about 3.5 days, theflask was evacuated and purged with nitrogen three times. The resultingmixture was filtered through 0.2-μm syringe filter and rinsed with IPA(2×4.0 mL). The colorless clear filtrate was then combined and the crudeproduct/IPA solution was used for the next reaction (assuming 100% yieldwithout purification).

Example 5 Preparation of ER-819302

The quantity of ER-819120 (mol) was calculated based on the startingER-819059 of the previous step.

To the 50-mL flask containing ER-819120 in IPA (2.52 g equivalent ofER-8189120, 5.87 mmol, ˜31.5 mL) was added di-tert-butyldicarbonate(1.30 g, 5.96 mmol) in one portion under a nitrogen atmosphere withstirring. The reaction was monitored by HPLC (Sample preparation: Sample15 μL, and add to 1.0 mL MeCN) and TLC (TLC, silica gel F254; mobilephase, MeOH/CH₂Cl₂/NH₄OH=10/89/1; visualization, anisaldehyde solutionor ninhydrin solution). TLC detected only minor ER-819120 spot. Moredi-tert-butyldicarbonate (0.20 g, 0.92 mmol) was added. No improvementwas noticed by TLC analysis. Solvent evaporation (29-32° C., 10 torr)gave a colorless clear oil (3.21 g).

A Biotage 12M silica gel (8.99 g, void volume=11.3 mL) cartridge wasconditioned with TBME/heptane=1/1 (36 mL, 3 v.v.). The flow rate wasadjusted to ˜12 mL/min. After dissolving the crude product (3.35 g) inTBME/heptane=1/1 (12 mL), it was loaded onto the cartridge, and elutedwith TBME/heptane=1/1 (70 mL). Fifteen fractions (˜6 mL each) werecollected and analyzed (TLC, silica gel F254; mobile phase,TBME/heptane=1/1; visualization, UV and anisaldehyde solution). Productfrom fractions (3-15) were combined. Solvent evaporation (29-32° C., 8.3torr) gave a colorless clear oil (3.03 g).

Example 6 Alternative Preparation of ER-819302

To ER-819059 (1.970 Kg, 3.82 mol) divided equally into two clean 12-Lreactors with isopropanol (IPA) (4.647 Kg each) was added 10% Pd/C (99g=flask 1, 102 g=flask 2). The flasks were evacuated (−0.79 bar) thenpurged with hydrogen (0.05 bar) three times while stirring. Thereactions were maintained under a hydrogen atmosphere (0.04 bar) of roomtemperature for 7 days and 16 hours after which time the reactions wereevacuated (house vacuum) and purged with nitrogen three times to removeall traces of excess hydrogen followed by cooling to 0° C. under anitrogen atmosphere.

In two separate flasks, di-tert-butyldicarbonate (434 g, 1.99 mol; and438 g, 2.01 mol, respectively) was dissolved in anhydrous THF (203 geach) under nitrogen atmosphere. Into each of the flasks, was addedcooled ER-819120 reaction mixtures over a 5-minute period. Anhydrous THF(40 g each) was used to rinse the residual reagents into the reactionmixture. The reactions were found to be exothermic (4.5 to 10.1° C.) andgassing was observed. The reactions were warmed to room temperature andcontinued to stir overnight. The completed reactions were combined andfiltered over Celite 545 (1.143 Kg, packed on a Fisher P5 24 cm filter)and rinsed with IPA (3.091 Kg) under a nitrogen blanket, The residue inthe reactors was rinsed and filtered with IPA (1.417 Kg) followed byrinsing the filter pad with IPA (13.46 Kg). The filter bed was rinsedtwo additional times with IPA (4.573 Kg and 6.360 Kg, respectively).

Concentration of the combined filtrates followed by azeotroping withheptane (7.529 Kg) gave a clear, colorless oil (2.452 Kg; 70.57 area %purity). The crude product was divided evenly into four portionspurification.

The crude ER-819302 (611 g, 1 wt) was dissolved in heptane (613 g, 1wt), and loaded onto a Biotage 150L silica gel cartridge [(5.620 Kg)conditioned with MTBE (10.48 Kg), and then with heptane (15.51 Kg) usinga flow rate of 700 mL/min]. Heptane (340 g, 0.56 wt) was used to rinsethe residual ER-819302 onto the column. The column was eluted with 15%MTBE/heptane (7.102 Kg/36.994 Kg), and then MTBE (15.72 Kg) wherefractions of approximately 3 L/each were collected. The remaining crudeER-819302 was separately chromatographed in three equal portions usingthe same method. The combined desired fractions from four purificationswere concentrated and azeotroped with heptane (0.5 Kg) to dryness, toprovide ER-819302 as a clear colorless oil (1.9135 Kg; 94.6% yield in97.86 area % purity).

Analytical Data for ER-819302-00

¹H-NMR (CDCl₃) δ 0.89 (t, J=6.9 Hz, 6H), 1.2-1.3 (m, 26H), 1.46 (s, 9H),1.5-1.7 (m, 4H), 1.7-1.8 (m, 1H), (m, 1H), 2.30 (t, J=7.6 Hz, 6H),3.3-3.4 (m, 1H), 3.48 (td, J=6.9, 9.6 Hz, 1H), 3.5-3.6 (m, 2H), 3.69(td, J=6.1, 7.1 Hz, 1H), 3.76 (d, J=8.2 Hz, 2H), 5.0-5.1 (m, 1H), 5.2(bs, 1H).

MS-APESI (M+Na) Calcd for C₃₀H₅₉NNaO₆: 552.42 Found: 552.52

KF=0.30%

Heptane=6034 ppm; MTBE=not detected.

Example 7 Preparation of ER-819344

Preparation 1:

To a stirred solution of diisopropylamine (22.4 mL, 0.160 mol) inanhydrous CH₂Cl₂ (200 mL) under a nitrogen atmosphere at roomtemperature was added pyridinium trifluoroacetate (30.9 g, 0.160 mol) inone portion providing a slight exotherm. Once the reaction mixturereturned to room temperature allyl tetraisopropylphosphorodiamidite(51.1 mL, 0.160 mol) was added followed by stirring for 10 minutes.ER-819302 (84.4 g, 0.159 mol), was azeotroped to dryness several timesusing anhydrous CH₂Cl₂ (300 mL) until the water content was determinedto be less than 60 ppm. After dissolving ER-819302 in dichloromethane(300 mL), the solution was slowly added to the above pyridinium reactionmixture maintaining the reaction temperature between 20 to 30° C.followed by rinsing the residue from the reagent vessel with additionaldichloromethane (100 mL).

When the formation of the reaction intermediate ER-820116 was complete(2 hours), the reaction mixture was cooled to 0° C. and followed by adropwise addition of acetic acid (18.2 mL, 0.320 mol) maintaining thereaction temperature between 0 to 15° C. Pyridinium trifluoroacetate (11g, 0.056 mol) was added to the reaction mixture and the resultingreaction was stirred for 10 minutes immediately after which timeER-222581 (46.5 g, 0.164 mol) was added in one portion. The reactionmixture was stirred at room temperature for 2 hours, then the mixturewas cooled 0° C. and 30-wt. % hydrogen peroxide in water (49 mL, 0.480mol) was added dropwise maintaining the final reaction temperaturebetween 0 to 10° C. (strong initial exothermic). The reaction mixturewas warmed up to room temperature and stirred for an additional 30minutes after which time the reaction mixture was cooled to 0° C. Thefinal reaction mixture was quenched with a slow addition of 10 wt. %aqueous sodium bisulfite (3.5 L) at an addition rate maintainingreaction temperature between 0 to 10° C. The quenched reaction wasallowed to warm to room temperature and stirred for until a negativeperoxide test for both ensuing layers (30 minutes).

The resultant mixture was diluted with MTBE (2.0 L), stirred for 10minutes and then transferred into a workup vessel. The layers wereseparated and the organic layer was washed one time each with 5% aqueousNaHCO₃ (2.0 L) and a 1:1 mixture of brine in water. The combined aqueouslayers were back extracted with MTBE (1 L). The combined organic layerswere dried over anhydrous sodium sulfate (100 g), filtered andconcentrated, and azeotroped to dryness with MTBE to provide ER-819344(146.6 g, 97% yield, 97% pure by HPLC).

Analytical Data for ER-819344

¹H-NMR (CDCl₃) δ 0.85-0.90 (m, 6H), 1.20-1.36 (m, 26H), 1.40-1.65 (m,4H), 1.44 (s, 9H), 1.70-1.83 (m, 2H), 2.27 (t, J=7.6 Hz, 2H), 3.37-3.57(m, 6H), 3.90-4.00 (m, 1H), 4.03-4.10 (m, 1H), 4.11-4.24 (m, 3H),4.35-4.40 (m, 2H), 4.50-4.60 (m, 2H), 4.94-5.0 (m, 1H), 5.05-5.15 (m,1H), 5.22-5.27 (m, 1H), 5.30-5.40 (m, 1H), 5.85-6.0 (m, 2H), 6.01-6.05(m, 1H), 7.30 (dd, J=7.3, 7.8 Hz, 2H), 7.40 (dd, J=7.3, 7.8 Hz, 2H),7.61 (d, J=7.8 Hz, 2H), 7.76 (d, J=7.3 Hz, 2H).

³¹P-NMR (CDCl₃, not calibrated) 0.172, 0.564 (two diastereomers)

MS-APESI (M+Na) Calcd for C₅₀H₇₉N₂NaO₁₁P: 937.53 Found: 937.65.

Preparation 2:

ER-819302 (1 wt.) was dissolved in anhydrous dichloromethane (3 vol.).If the total amount of water is greater than or equal to 0.7 mol. % ofER-819302, as determined by K_(f), then the water content is lowered toa satisfactory level by chasing the water with an evaporating solvent.

Anhydrous dichloromethane (2 vol.) was charged to a dry reactor,followed by diisopropyl amine and pyridinium trifluoroacetate (1 eq.)(in bath before added to control exotherm). The solution was stirred andthe temperature adjusted in a bath to 20 to 25° C. Allyltetraisopropylphosphorodiamidite (1 eq.) was then charged to thesolution followed by stirring for five minutes. The dichloromethanesolution of ER-819302 was then added to the solution at controlledaddition rate while maintaining reaction temperature below 30° C. (rinsewith 1 vol. dichloromethane). The reaction progress was monitored by TLC(MTBE/Heptane/Et3N=40/60/1) and HPLC (TM, samples were prepared bywithdrawing a 30-ul reaction mixture and diluting it with 1 mlacetonitrile). The reaction is complete when the ER-819302:ER-820116ratio is greater than 95:5, which usually occurs in 2 hours. Afterformation of the intermediate ER-82 0116, the reaction mixture wascooled to 0-10° C. and charged with acetic acid at an addition rate thatmaintains the reaction temperature below 15° C.

Pyridinium trifluoroacetate (0.4 eq.) was charged into reaction mixture,followed by ER-222581 (1 eq.). The mixture was stirred at roomtemperature for approximately 20-30 minutes until the white suspensionbecame a clear solution. The reaction progress was monitored by TLC(MTBE/Heptane/Et3N=40/60/1) and HPLC (TM, samples were prepared bywithdrawing a 30-ul reaction mixture and diluting it with 1 mlacetonitrile). After the reaction was complete, about 1.5 or 2 h, thereaction mixture was cooled to −5 to 0° C.

30 wt. % Hydrogen peroxide (3 eq.) in water was then charged into thereaction mixture while maintaining reaction temperature below 5° C. Thereaction mixture was allowed to warm up to room temperature, and thenstirred for 30 minutes. It was cooled back to −5 to 0° C., and 10 wt. %aqueous sodium bisulfite solution was charged while maintaining areaction temperature below 5° C. After charging the bisulfite solution,the reaction mixture was again allowed to warm up to room temperature,and then stirred for 30 minutes. Stirring was continued until thereaction mixture indicated a negative result on a peroxide testingstrip.

Methyl t-butyl ether was charged into the reaction and stirred for 10minutes. The reaction mixture was then transferred into a workup vesseland the layers allowed to separate. If the aqueous layer was shown tocontain product, it was back extracted with methyl t-butyl ether. Theorganic layers were washed with 5% aqueous sodium bicarbonate followedby a solution of half brine (if the aqueous layer is hazy, backextraction with methyl t-butyl ether may be necessary), and the organicwas concentrated (if it became a milky oil, it was charged with MTBE andvacuum filtered). The crude ER-819344 was analyzed by HPLC and HNMR. Thelargest scale run produced 84.4 g ER-819302 with a 97% yield, asindicated by HPLC.

Example 8 Preparation of ER-819385-00

To a stirred solution of ER-819344 (1.56 g, 1.70 mmol) in THF (1.5 mL)at room temperature was added 2.0 M dimethylamine in THF (8.5 mL, 17.0mmol) and the reaction mixture was stirred for 2 h. The completedreaction mixture was concentrated and the crude product was azeotropedto dryness two times with MTBE (15 mL). The resultant product wasdissolved in MTBE (30 mL), and washed with brine (7.5 mL). The finalorganic layer was concentrated down to dryness, azeotroped with one timewith MTBE (15 mL) to provide the desired, somewhat unstable ER-819385that was used in the next step without further purification.

Preparation 2:

ER-819344-00 (1.56 g, 1.70 mmol) was dissolved in THF (1.5 mL), andadded to a commercial solution of dimethylamine in THF (2.0 M, 8.5 mL)at room temperature and stirred for 2 h. A TLC analysis was conductedwhich showed complete consumption of ER-819344. U.V. lamp andp-anisaldehyde stain were used as visualization techniques. Thevolatiles were then removed through rotary evaporation techniques. Thecrude product was azeotroped with MTBE (2×15 mL), after which it wasdissolved in MTBE (30 mL) and washed with brine (7.5 mL to remove anyresidual low MW amines (for example, dimethylamine, ethanolamine). Noemulsions were obtained, as the layers separated easily. The pH of theaqueous layer after this wash was ˜10. The organic layer wasconcentrated down to dryness and azeotroped with MTBE (1×15 mL) toprovide desired amine monomer ER-819385-Oft

Example 9 Preparation of ER-819409-00

Preparation 1:

To a stirred solution of crude ER-819385 (calc. 1.18 g, 1.70 mmol) inCH₂Cl₂ (15 mL) was added a saturated solution of NaHCO₃ (12 mL). Theresulting mixture was cooled to 0° C. followed by a dropwise addition of20% phosgene in toluene (465 μL, 0.935 mmol). The reaction was allowedto warm up to room temperature, stirred for 1 hour, then was cooled to0° C. before the addition of a second portion of 20% phosgene solution(210 μL, 0.425 mmol). The final reaction mixture was warmed to roomtemperature, stirred overnight. Water (15 mL) was added. After stirringfor an additional 30 minutes the quenched reaction was transferred intoa separatory funnel and the layers allowed to separate for 45 min. Theaqueous layer was extracted with CH₂Cl₂ (30 mL) and the combined organiclayers were concentrated to dryness. The crude oil was azeotroped todryness with MTBE/ethyl acetate (EtOAc) (1:1, 50 mL) dissolved inEtOAc/heptane (1:1, 50 mL) and filtered on a fritted funnel to removesalts. The crude product was purified over silica gel (12 g) eluted with2.5% MeOH/Ethyl acetate (50 mL), with 3.8% MeOH/Ethyl acetate (50 mL),and finally with 5.6% MeOH/Ethyl acetate (100 mL) to provide ER-819409(984 mg, 82% yield) as a clear, colorless oil.

Preparation 2:

ER-819385-00 (1.70 mmol, crude material) was dissolved in CH₂Cl₂ (15 mL)and added to a saturated solution of NaHCO₃ (12 mL). The resultingmixture was cooled to 0 C and added dropwise to a commercial solution ofphosgene in toluene (465 uL, 0.55 equiv.). The reaction was allowed towarm up to room temperature under stirring for 1 h. TLC (10%MeOH/CH₂Cl₂) and p-anisaldehyde analyses were used to visualize reactionproducts and revealed that a large amount of starting material was stillpresent. The reaction was therefore cooled to 0° C. for a secondaddition of a 20% phosgene solution (210 uL, 0.25 equiv.). After theaddition, the temperature of the reaction was allowed to slowly warm upto room temperature. TLC analysis 1 h later still showed startingmaterial but indicated no signs of decomposition. The reaction was thenallowed to sit overnight while being stirred. TLC analysis the next daystill indicated presence of starting material., but also revealed theoccurrence of base-line decomposition. Water was added (15 mL) to thereaction product at room temperature and stirred for 30 min. The mixturewas transferred to a separatory funnel and the layers were allowed tostand for 45 min before they were separated. The aqueous phase wasextracted with CH₂Cl₂ (30 mL) and the organic layers were combined andconcentrated to dryness. TLC analyses of the aqueous and the combinedorganic layers did not indicate the presence of amine starting material,indicating that the reaction went to completion during the work up.

The crude oil was azeotroped with MTBE/EA, re-dissolved in ˜50 mlEA/heptane at a 1:1 ratio, and filtered on a fitted funnel to removesalts. This material was purified on a SiO₂ column (50% EA/n-heptane,100% EA, then 5-10% MeOH/EA) to provide 984 mg of the desired ureaER-819409-00, present as a colorless oil. This represents an 82%combined yield since the Fmoc deprotection. The mass balance wasbase-line material that formed during the overnight stirring of theurea-formation stage.

Example 10 Dihydroxy Urea Synthesis of ER-819409-00

To a stirred solution of pyridinium trifluoroacetate (108 g, 0.533 mol)in anhydrous acetonitrile (348 g) was added diisopropylamine (78.7 mL,0.561 mol) at a rate to maintain the reaction temperature below 30° C.After allowing the reaction to cool to room temperature, allyltetraisopropylphosphorodiamidite (179 mL, 0.561 mol) was added (slightendotherm then exotherm was observed) followed by stirring for anadditional 10 minutes. Subsequently ER-819302 (283.2 g, 0.5345 mol;pre-chased with 800 mL of Heptane) in anhydrous acetonitrile (453 mL)was added at an addition rate to maintain reaction temperature between20 and 30° C. After stirring for an additional 30 minutes the reactionmixture was cooled to 0° C. followed by the slow addition of acetic acid(64 mL, 1.1 mol) while maintaining reaction temperature below 25° C. Thereaction mixture was allowed to equilibrate at room temperature.Pyridinium trifluoroacetate (103 g, 0.533 mol) in acetonitrile (200 mL)was added to the reaction mixture. Immediately after the addition ofpyridinium trifluoroacetate was added ER-812978 (40 g, 0.27 mol)followed by an acetonitrile rinse (50 mL). The reaction mixture wasstirred for 18 h at room temperature after which time it was cooled to0° C. followed by the slow addition of 30 wt. % hydrogen peroxide inwater (140 mL, 1.37 mol) maintaining reaction temperature between 0 to24° C. (initially a strong exotherm). The final reaction mixture wasstirred for an additional 30 minutes followed by addition of 20-wt %aqueous sodium bisulfate solution (3000 g) at a rate maintainingreaction temperature between 0 and 18° C. The reaction mixture waswarmed up to room temperature and stirred until peroxide testingprovided a negative result.

The resultant reaction mixture was diluted with MTBE (3000 mL) in aworkup vessel and stirred for 15 minutes. After separation of thelayers, the organic layer was washed with 10% aqueous sodium bicarbonate(NaHCO₃) (3500 mL) and then with 30% aqueous NaCl solution (2000 mL).The brine layer was back-extracted with MTBE (3000 mL) three times. Thecombined organic layers were concentrated, and chased withTBME/Heptane=1/1 (1.4 L) twice. The residue was dissolved in MTBE (735g), and the suspension filtered through a Celite pad (150 g) followed bysubsequent three MTBE (300 mL) washings of the vessel and filter pad.The filtrate was concentrated to dryness to give 363.4 g of slightlycloudy oil. The crude ER-819302 was dissolved loaded onto apre-conditioned silica gel cartridge [Biotage 150L (5.62 Kg, voidvolume=7.07 L) cartridge conditioned with MTBE/Heptane=7/3 (15 Kg)] withTBME/Heptane=7/3 (400 mL) using an adjusted flow rate of ˜800 mL/min.After loading, TBME/Heptane=7/3 (500 mL) was used to rinse residualER-819302 and the rinse loaded onto the cartridge. The cartridge waseluted with MTBE/Heptane=7/3 (26 Kg), and then withMTBE/Heptane/MeOH=70/25/5 (21.8 Kg/7.18 Kg/1.7 Kg). A total of 36fractions were collected during this process. The combined, productcontaining fractions were concentrated and azeotroped to dry usingheptane (8 L) followed by drying under house vacuum to give an oil (281g, 79%) at 92.69 area % purity.

Analytical Data for ER-820116

¹H-NMR (CDCl₃) δ 0.870 (m, 6H), 1.176 (d, J=8.0 Hz, 12H), 1.254-1.282(b, 28H), 1.428 (b, 9H), 1.528 (b, 2H), 1.603 (b, 2H), 1.790 (m, 2.265(t, J=7.0 Hz, 2H), 3.431 (m, 2H), 3.481 (m, 2H), 3.600 (m, 2H), 3.751(b, 2H), 3.847 (b, 1H), 3.847-4.208 (m, 2H), 4.950 (b, 1H), 5.126 (d,J=10 Hz, 1H), 5.286 (dd, J=17 Hz, J′=1.5 Hz, 1H), 5.898-5.989 (m, 1H)

³¹P-NMR (CDCl₃, calibrated) 148.449 and 148.347 (two diastereomers)

MS-APESI (M+H) Calcd for C₃₉H₇₈N₂O₇P: 717.55. Found: 717.66.

Analytical Data for ER-821843

³¹P-NMR (CDCl₃, calibrated) δ 139.832, 139.868, 140.170, 140.327 (4diastereomers)

MS-APESI (M+Na) Calcd for C₃₀H₅₉NNaO₆: 552.42 Found: No Mass data

Analytical Data for ER-819409

MeOH: not detected

MTBE: not detected

MeCN: 185 ppm

Heptane: 1718 ppm

¹H-NMR (CDCl₃) δ 0.88 (t, J=6.9 Hz, 12H), 1.20-1.37 (m, 52H), 1.44 (s,18H), 1.45-1.72 (m, 8H), 1.76-1.85 (m, 4H), 2.28 (t, J=7, 6 Hz, 4H),3.38-3.58 (m, 12H), 3.85-3.97 (m, 2H), 3.98-4.20 (m, 8H), 4.53-4.58 (m,4H), 4.95-5.0 (m, 2H), 5.18-5.28 (m, 2H), 5.26 (dd, 10.5 Hz, 2H), 5.37(dd, J=0.9, 16.9 Hz, 2H), 5.62-5.85 (m, 2H), 5.87-5.99 (m, 2H).

MS-APESI (M+Na) Calcd for C₇₁H₁₃₆N₄NaO₁₉P₂: 1433.92 Found: 1433.98.

Example 11 Preparation of ER-807284-00

Preparation 1:

To a stirred solution of ER-819409 (40.25 g, 28.51 mmol) in dry CH₂Cl₂(120 mL) under a nitrogen atmosphere was added slowly over a 10-minuteperiod a solution of methanesulfonic acid (13.8 g, 144 mmol) in CH₂Cl₂(140 mL) while maintaining the reaction temperature below 20° C. Thereaction mixture was warmed to 20° C. followed by stirring 15 hours whenthe intermediate reaction was determined to be complete. The resultingreaction mixture was cooled to 0° C. and diisopropylethylamine (27.5 mL,158 mmol) was added over a 5-minute period. After 5 minutes ofadditional stirring at 0° C.,1-(3-dimethylaminoproply)-3-ehylcarbodiimide hydrochloride (32.55 g, 170mmol) was added in one portion. The reaction mixture was stirred at 0°C. for 12 minutes followed by the addition of ER-028699 (20.6 g, 85.0mmol) in one portion. The resulting reaction mixture was stirred for 2hours at 0° C. followed by warming to room temperature for 30 minutes atwhich time the reaction was determined complete.

One fourth of the completed reaction mixture (105 g) was eluted onto apre-condition Biotage 75M silica gel cartridge [(351 g silica gel,conditioned with MTBE (1 L) and then with CH₂Cl₂ (2 L)] with a flow rateadjusted to 150-200 mL/min. The column was eluted sequentially with 1%ethanol (EtOH)/CH₂Cl₂ (900 mL), with 3% EtOH/CH₂Cl₂ (900 mL) and finallywith 6% EtOH/CH₂Cl₂ (2250 mL) while collecting ˜150 mL/fractions. Thedesired product containing fractions were combined concentrated (housevacuum, 30-35° C.) and azeotroped three times with heptane (100 mL) toprovide 8.8 g of ER-807285. The remainder of the completed reactions waspurified in a similar manner to provide a total of 35.2 g (74.5% yield).

Two additional experimental procedures are described below. They differat the work-up stage. The first one involves a standard quench and theother one uses a reverse quench. TLC analyses were performed withNH₄OH/MeOH/CH₂Cl₂ 1:9:90. TLC plates were charred with p-anisaldehydestain to visualized starting material and reaction products.

Preparation 2:

ER-819409-00 (995 mg, 0.705 mmol) was dissolved in CH₂Cl₂ (7.8 mL). TFA(1.4 mL) was added to this mixture at room temperature over 1-2 min. Thereaction mixture was then stirred 4 h at room temperature.

After stirring, the reaction mixture was cooled with an ice bath and asaturated aqueous solution of NaHCO₃ (16.0 mL) was added over 25 min.The highest temperature recorded during the neutralization was ˜8° C.,with an average temperature of 4° C. The resulting mixture was stirredan additional 45 min during which time the internal temperature wasallowed to slowly warm up from 4° C. to room temperature. The mixturewas transferred to a separation funnel and CH₂Cl₂ was added (11.0 mL).The layers were allowed to stand for 35 min before being separated. Theaqueous layer, which had a pH of 8˜8.5, was extracted with CH₂Cl₂ (5.0mL), The organic layers were combined and washed with 10 mL of a salinesolution that was prepared by mixing a saturated brine solution withwater in a 3:1 ratio. The layers were allowed to stand for 20 min beforebeing separated. The organic layer was then stored in the freezer (−20°C.) overnight.

The next morning, the organic layer was removed from the freezer andallowed to warm up to room temperature. It was dried using Na₂SO₄,filtered on a fritted-funnel, and concentrated down to dryness. Theresulting oil was re-dissolved in CH₂Cl₂ (8.0 mL) and filtered on acotton plug in order to remove any salt residues. The resulting materialwas concentrated to dryness to produce the colorless oil ER-807284-00(727 mg, 85% mass recovery). An additional CH₂Cl₂ extraction provided anextra 99.0 mg of desired material bringing the mass recovery to 96.8%.No purification was necessary.

Analytical Data for ER-807284

¹H-NMR (CDCl₃) δ 0.85-0.95 (m, 12H), 1.20-1.35 (m, 52H), 1.45-1.65 (m,8H), 1.70-1.85 (m, 4H), 2.25-2.65 (bs, 4H), 2.28 (t, J=7, 6 Hz, 4H),3.20-3.27 (m, 211), 3.30-3.60 (m, 12H), 3.98-4.22 (m, 10H), 4.50-4.60(m, 4H), 4.95-5.05 (m, 2H), 5.27 (dd, J=0.9, 10.5 Hz, 2H), 5.38 (dd,J=0.9, 16.9 Hz, 2H), 5.90-6.0 (m, 2H).

MS-APESI (M+H) Calcd for C₆₁H₁₂₁N₄O₁₅P₂: 1211.83 Found: 1211.97.

Preparation 3:

An appropriate sized reactor was charged with containing CH₂Cl₂ (22.3mL). ER-819409-00 (2.85 g, 2.01 mmol) was added and dissolved in theCH₂Cl₂.TFA (4.0 mL) was added over 1 min at r.t. The reaction mixturewas stirred 4.5 h at room temperature.

Work-Up: (Reverse quench): The reaction mixture was transferred via aTeflon canula over 1-2 min to a saturated solution of NaHCO₃ cooled to0° C. Slight warming was observed, max exotherm ˜4° C. The reactionflask was rinsed with CH₂Cl₂ (4×2.5 mL) and the washings added to thesolution. The cooling device was removed and the temperature was allowedto warm up to room temperature over 45 min. Additional CH₂Cl₂ (22 mL)was added and the mixture was transferred to a separatory funnel. Themixture was allowed to stand for 20 min before separation. The aqueouslayer was extracted with CH₂Cl₂ (22 mL) and the organic layers combined.The combined organic layers were washed with a saline solution,saturated brine/H₂O (3:1 ratio) (40 mL). The resulting mixture wasallowed to stand for 30 min while the layers slowly separated. Thelayers were separated. The organic layer remained cloudy. The organiclayer was stored in a −20° C. freezer overnight. It was then allowed towarm up to room temperature, dried with Na₂SO₄, filtered on a frittedfunnel and concentrated to dryness. The aqueous brine solution was alsoback-extracted with CH₂Cl₂ (22 mL) to recover additional material.Proton and fluorine NMR spectra revealed that these two crops ofmaterial were contaminated by TFA salt forms. pH analysis of the NaHCO₃layer revealed that its pH was ˜7 (not sufficiently high (8-8.5) tocleanly give the free base ER-807284-00). The two crops obtained abovewere combined and the work up was repeated by dissolving the organicmaterial in CH₂Cl₂ (50 mL), The combined solution was transferred to a250 mL three-neck-round bottom flask equipped with a mechanical stirringdevice. A saturated aqueous solution of NaHCO₃ (50 mL) was added and theresulting mixture was stirred 45 min at room temperature. The content ofthe reactor was transferred to a 250 mL sepratory funnel. The reactorwas rinsed with CH₂Cl₂ (total 25 mL). The mixture was allowed to standfor ˜1 h and emulsions were observed. The organic and aqueous layerswere separated and the aqueous layer was back-extracted with CH₂Cl₂ (30mL). The resulting organic layers were combined, washed with saturatedbrine (25 mL). Even after standinf for 1 h, the organic layer remainedcloudy after which time the organic and aqueous layers were separated.The organic layer was dried with Na₂SO₄, filtered on a fritted-funnel.The filtrate was cloudy. The brine solution was back-extracted withCH₂Cl₂ (25 mL), The material from the CH₂Cl₂ layer was combined with theother material after a similar drying procedure. The resulting combinedorganic filtrates were concentrated down to dryness, azeotroped withMTBE (2×25 mL), re-dissolved in CH₂Cl₂ (10 mL) and filtered on a Celite(3 mL) plug located in 10 mL syringe. The tip of that syringe was alsoequipped with a filtration device to catch small particles. The filtratewas concentrated to dryness and NMR spectroscopy revealed that thediamine ER-807284-00 was obtained and was free of TFA salts. The massrecovery was over 95%. The reaction was clean by TLC. The work up had tobe repeated to cleanly generate the freebase and some degradation becameapparent by TLC. pH control may improve this procedure.

Example 12 Preparation of ER-807285-00

Preparation 1—EDC/HOBT:

An appropriately sized inert reactor was charged with ER-807284-00 (1.0equivalent) and anhydrous methylene chloride (8.41 weights). The reactorwas then charged with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (2equivalents) followed by 1-hydroxybenzotriazole (0.18 equivalent). Thereactor was then charged with 3-oxo-tetradecanoic acid (2.2 equivalents)in three equal portions, letting the reaction mixture stir for 10 minbetween each charge keeping T_(internal) at 15-20° C. The reaction wasmonitored by TLC for complete consumption of ER-807284. When thereaction was determined to be complete (typically after 1 h), processwater (5 weights) was charged to the reactor. The mixture was allowed tostir for 20 minutes and then allowed to separate for 20 minutes. Theorganic layer was set aside. The aqueous layer was back extracted in theabove manner with ethyl acetate two times (2×6 weights). All organiclayers were then combined, charged with sodium sulfate (8 weights) andallowed to stand 15 min to absorb moisture. The organics were filteredand the cake was washed with ethyl acetate until a negative result forER-807285 was obtained. The filtrates were concentrated in vacuo (˜50torr at 28-35° C.) affording ER-807285. That material was purified bysilica gel chromatography using 3%-6% EtOH/CH₂Cl₂. Fractions rich indesired material were combined and concentrated down by rotoevaporationand dried with IVAC pump (0.2 torr) for 2 h. The yield was 50% of acolorless oil, ER-807285.

Preparation 2—EDC/DMF:

ER-807284-00 (208 mg, 0.172 mmol.) was dissolved in DMF (2.1 mL) in anappropriate sized reactor and EDC (263 mg, 1.37 mmol) added. The mixturewas cooled to 0° C. and 3-oxotetradecanoic acid (166 mg, 0.686 mmole)dissolved in DMF (1.4 mL) was added dropwise over 30 sec. The resultingreaction mixture was stirred at 0° C. for 10 min while warming to roomtemperature. The reaction was monitored by TLC (7.5% MeOH/CH₂Cl₂:p-anisaldehyde stain for the starting materials and products. Thereaction was quenched ˜3 h later at 0° C. by the addition of a saturatedsolution of NaHCO₃ (8.0 mL), H₂O (4.0 mL) and MTBE/n-heptane 1:1 (10mL). The reaction mixture was transferred to a separatory funnel. Asmall amount of MTBE/n-heptane 1:1 was used to rinse the reactor andthen combined with the reaction mixture. The reaction mixture wasallowed to for 20-30 min after which time the organic and aqueous layerswere separated. The total volume of the organic layer was ˜35 mL.Analysis of the aqueous layer by TLC showed a small amount of DMF. Asecond extraction of aqueous layer was not needed. The organic layer waswashed with brine (4.0 mL) and then was allowed to stand 15 min. A fastseparation was observed with no emulsions. The organic and aqueouslayers were separated and the organic layer was evaporated to dryness toproduce crude ER-807285-00. The crude ER-807285 was purified on a SiO₂column using a: 3-6% EtOH/CH₂Cl₂ solvent system. The yield was 53%, (151mg of ER-807285), 88% pure by HPLC.

Preparation 3—HBTU/Hunig's base/DMF:

ER-807284-00 (232 mg, 0.191 mmol,) was dissolved in DMF (2.5 mL). Thereactor was cooled to 0 C. HBTU (218 mg, 0.574 mmol,) and3-oxotetradecanoic acid (139 mg, 0.574 mmole) were added. This wasfollowed by Hunig's base (106 uL, 0.612 mmol) dropwise over 30 sec. Thereaction mixture was stirred 20 min at 0 C and became milky after ˜10min. The reaction mixture was allowed to warm up to room temperature.Stirring was continued over 4 h. TLC monitoring is difficult due to DMF.Accordingly the reaction time might be shorter. The reaction mixture wasdiluted with MTBE/n-heptane 1:1 (10 mL), transferred to a 60 mLseparatory funnel, and treated with an aqueous solution prepared bymixing citric acid 1.0 M (50 uL) and saturated sodium chloride (9.5 mL)at pH 3). Significant amounts of salts were formed and crashed out,clogging the funnel. Water (5.0 mL) was added to dissolve the salts butafter that, no phase separation was possible even after progressivelyadding MTBE (up to 15 mL). Ethyl acetate was then added taking up to 10mL to start restoring phase separation. The layers were allowed to stand˜30 min to achieve separation. The pH of the aqueous phase was adjustedto pH 5. The organic layer was washed again with 10 mL of dilute citricacid prepared as described above, resulting in a pH of 3. The layerswere separated. The organic layer was washed with a saturated solutionof NaHCO₃ (2×5 mL). The resulting aqueous and organic layers wereseparated and the organic layer was evaporated to dryness. The yield ofER-807285 was 45% (143 mg) with 91% purity by HPLC.

Example 13 Preparation of E6020

An appropriately sized inerted vessel was charged with ER807285 (1equivalent) in degassed THF (1.57 weight) under an argon stream. Asolution of tetrakis(triphenylphosphine)palladium (0) (0.03 weight),triphenylphosphine (0.03 weight) and phenylsilane (0.07 weight) intetrahydrofuran (2 weights) was charged in the reactor over 40 min(T_(internal) typically raise to ˜40-45° C.). The reaction was monitoredby TLC and HPLC for complete consumption of ER-804057. When the reactionwas determined to be complete (typically after <10 minutes), thereaction mix was purified by ion exchange chromatography. For moredetails about this purification, see example 14.

Example 14 Purification of E6020

A crude reaction mixture of 804057 free acid containing tetrakistriphenylphosphine palladium (0), triphenylphosphine, and phenylsilanewas loaded onto a Source 30Q ion-exchange column. Then, the non-bindingreactants were eluted away from the 804057 using methanol/THF/water(77.5/15/5). The sodium salt of 804057 (i.e., E6020) eluted from thecolumn by using an increasing linear gradient of sodium acetate thatstarts at 0 M and ends at 0.05 M. Impurities were removed during thischromatography.

An optional second purification may be desired. This chromatographystarts with the E6020/sodium acetate ion-exchange solution obtained fromthe previous chromatography. This was directly loaded onto a C-4Kromasil column and eluted with the isocratic buffer systemmethanol/THF/water/sodium acetate (77.5/15/5/0.05 M). The fractionscontaining product were combined for solid phase extraction.

Pure E6020 solutions were then diluted 50/50 with water and loaded ontothe C-4 Kromasil column. This was then eluted with water, a lineargradient from water to acetonitrile. This separated the salt and waterfrom pure E6020. Then, the product was eluted from the column usingmethanol. A solution of pure E6020 in methanol was obtained. This wasconcentrated to dryness on a rotary evaporator at 25 to 30° C. and fullhouse vacuum. The glassy product was lyophilized or treated with asolution of ethyl acetate/acetonitrile, which formed a white solid. Thiswas vacuum dried to give E6020.

Example 15 Characterization of Crystalline ER-806158,(R)-1-(((R)-4,5-dihydro-2-phenyloxazol-4-yl)methoxy)decan-3-ol

A portion of ER-806158 was re-dissolved in warm toluene until all thematerial dissolved, and was allowed to cool. This resulted in singlecrystals from which one was chosen to be used in this study. A colorlessblock crystal with dimensions 0.14×0.14×0.10 mm was mounted on a glassfiber using very small amount of paratone oil.

A. Single Crystal X-Ray Diffraction

Data were collected using a Bruker SMART APEX CCD (charge coupleddevice) based diffractometer equipped with an Oxford Cryostreamlow-temperature apparatus operating at 193 K, Data were measured usingomega scans of 0.3° per frame for 30 seconds, such that a hemisphere wascollected. A total of 1271 frames were collected with a maximumresolution of 0.76 Å. The first 50 frames were recollected at the end ofdata collection to monitor for decay. Cell parameters were retrievedusing SMART software (SMART V 5.625 (NT) Software for the CCD DetectorSystem; Bruker Analytical X-ray Systems, Madison, Wis. (2001)). andrefined using SAINT on all observed reflections. Data reduction wasperformed using the SAINT software (SAINT V 6.22 (NT) Software for theCCD Detector System Bruker Analytical X-ray Systems, Madison, Wis.(2001). which corrects for Lp and decay. The structures were solved bythe direct method using the SHELXS-97 program (Sheldrick, G. M.SHELXS-90, Program for the Solution of Crystal Structure, University ofGottingen, Germany, 1990.) and refined by least squares method on F²,SHELXL-97, (Sheldrick, G. M. SHELXL-97, Program for the Refinement ofCrystal Structure, University of Gottingen, Germany, 1997.) incorporatedin SHELXTL-PC V 6.10, (SHELXTL 6.1 (PC-Version), Program library forStructure Solution and Molecular Graphics; Bruker Analytical X-raySystems, Madison, Wis. (2000)).

The structure, shown in FIG. 1, was solved in the space group P1 (#1) byanalysis of systematic absences. All non-hydrogen atoms are refinedanisotropically. Hydrogens were found by difference Fouier methods andrefined isotropically. The crystal used for the diffraction study showedno decomposition during data collection. All drawing are done at 50%ellipsoids. FIG. 2 is the packing diagram along the a-axis which showsthe best diagram of the hydrogen bonding within the crystal, dottedlines.

TABLE 1 Crystal data and structure refinement. Wavelength 0.71073 ÅCrystal system Triclinic Space group P1 Unit cell dimensions a =4.6047(11) Å α = 106.008(4)°. b = 8.1161(19) Å β = 95.604(4)°. c =13.579(3) Å γ = 98.696(4)°. Volume 477.0(2) Å³ Z   1 Density(calculated) 1.161 Mg/m³ Absorption coefficient 0.077 mm⁻¹ F(000)  182Crystal size 0.14 × 0.14 × 0.10 mm³ Theta range for data collection 1.58to 27.93°. Index ranges −6 <= h <= 6, −10 <= k <= 7, −12 <= l <= 17Reflections collected 3293 Independent reflections 2663 [R(int) =0.0431] Completeness to theta = 27.93° 98.3% Absorption correction NoneMax. and min, transmission 0.9924 and 0.9893 Refinement methodFull-matrix least-squares on F² Data/restraints/parameters 2663/3/342Goodness-of-fit on F²   1.006 Final R indices [I > 2sigma(I)] R1 =0.0474, wR2 = 0.1231 R indices (all data) R1 = 0.0527, wR2 = 0.1275Absolute structure parameter 0.0(16) Largest diff. peak and hole 0.252and −0.252 e · Å⁻³

B. Powder X-Ray Diffraction

Using a quartz plate, on a Scintag Diffractometer, data was run undernormal powder diffraction conditions, with 2-theta range of 5-70degrees, using copper radiation and analyzed under the conditions shownin Table 2. No background correction was applied. FIG. 3 shows the PXRDpattern of crystalline ER-896158. Characteristic peaks for the PXRDpattern of crystalline ER-896158 are listed in Table 3.

TABLE 2 Measurement conditions X-ray diffractometer: Scintag Target: Cuγ Detector: Lithium Drifted Diode Tube voltage: 40 kV Tube current: 30mA Slit: DS 1.0, RS 0.3 mm, SS 2 mm tube, 0.5 m detector Scan speed:1°/min StepISampling: 0.02″ Scan range: 5 to 70″ Sample holder: Quartzholder (25 mm × 25 mm) Goniometer: Theta-Theta, fixed horizontal mount,goniometer Filter: Electronic Monochromator: not used

TABLE 3 Characteristic Powder X-ray Diffraction Peaks (2Θ ± 0.2 2Θ) 6.911.9 13.6 19.5 19.7 20.2 20.5 21.7 23.3 24.2 25.2 25.4 26.5 27.4 34.441.6 48.9 55.2 58.6

C. Characterization of Crystalline ER-806158 by DSC.

Solid-state characterization of crystalline ER-806158 was determined byDifferential Scanning calorimetry (DSC, capillary technique). Using a5.17000 mg sample of crystalline ER-806158, the DSC was run on a 2920DSC V2.5F calorimeter heating to 200° C. at 10° C./min with an aluminapan under a nitrogen purge of 50 mL/min. FIG. 4 shows the thermograms ofcrystalline ER-806158 melted at 27° C. (onset temp.) absorbing+29.2cal/g in the presence of nitrogen. A melt preceded by an exothermicevent was observed during a reheat of the sample, which indicates thisER-806158 may be stable to 200° C. in the liquid phase.

D. Infrared Spectrum of Crystalline ER-806158

The FTIR absorption spectrum of crystalline ER-806158 was recorded forthe neat crystalline powder. The IR absorption spectrum of crystallineER-806158 is shown in FIG. 5.

1.-32. (canceled)
 33. Crystalline ER-806158,(R)-1-(((R)-4,5-dihydro-2-phenyloxazol-4-yl)methoxy)decan-3-ol having atriclinic crystal system and a P1 space group.
 34. CrystallineER-806158,(R)-1-(((R)-4,5-dihydro-2-phenyloxazol-4-yl)methoxy)decan-3-ol, of claim33, characterized by crystal lattice parameters of: a = 4.6047(11) Å α =106.008(4)° b = 8.1161(19) Å β = 95.604(4)° c = 13.579(3) Å γ =98.696(4)° Volume = 477.0(2) Å³ and Z = 1.